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University of Szeged
Pharmaceutical Analysis Practicals
Edited by:
György Dombi
Gerda Szakonyi
Authors:
György Dombi
Éva Kalmár
Gerda Szakonyi
Henriett Diána Szűcs
Reviewed by:
Krisztina Novák-Takács
Szeged, 2015
This work is supported by the European Union, co-financed by the European Social Fund,
within the framework of "Coordinated, practice-oriented, student-friendly modernization of
biomedical education in three Hungarian universities (Pécs, Debrecen, Szeged), with focus on
the strengthening of international competitiveness" TÁMOP-4.1.1.C-13/1/KONV-2014-0001
project.
The curriculum can not be sold in any form!
TABLE OF CONTENTS
CONDUCTOMETRY ....................................................................................................................... 3 CONDUCTOMETRIC TITRATION OF CARBOXYLIC ACIDS .............................................................. 7 ANALYSIS OF ACETYLSALICYLIC ACID ....................................................................................... 8 ANALYSIS OF BENZOIC ACID ........................................................................................................ 9 POTENTIOMETRIC (pH-METRIC) TITRATIONS ............................................................................ 10 HOW TO USE THE GLASS ELECTRODE ....................................................................................... 15 POTENTIOMETRIC TITRATION .................................................................................................... 15 EVALUATION OF THE MEASUREMENT ....................................................................................... 15 QUANTITATIVE ASSAYS BY TITRATION WITH ALKALINE SOLUTIONS........................................ 16 DINATRII PHOSPHAS DIHYDRICUS ............................................................................................. 17 NATRII DIHYDROGENOPHOSPHAS DIHYDRICUS ......................................................................... 18 CHININI HYDROCHLORIDUM ..................................................................................................... 19 UNGUENTUM AD VULNERA ....................................................................................................... 21 SPECTROPHOTOMETRY .............................................................................................................. 23 PULVIS CHINACISALIS CUM VITAMINO C .................................................................................. 33 TABLETTA ASPIRINI 500 (ASPIRIN TABLET 500) ....................................................................... 37 SUPPOSITORIUM PARACETAMOLI 500 MG.................................................................................. 39 SPARSORIUM ANTISUDORICUM ................................................................................................. 41 SOLUTIO METRONIDAZOLI ........................................................................................................ 43 PULVIS CHOLAGOGUS ............................................................................................................... 44 DETERMINATION OF PROTEIN CONCENTRATION WITH THE BIURET REAGENT .......................... 47 ATOMIC ABSORPTION SPECTROMETRY ..................................................................................... 49 DETERMINATION OF MAGNESIUM CONTENT OF SPARSORIUM ANTISUDORICUM BY FLAME
ATOMIC ABSORPTION................................................................................................................ 53 DETERMINTION OF MAGNESIUM CONTENT OF PULVIS NEUTRACIDUS BY FLAME ATOMIC
ABSORPTION ............................................................................................................................. 54 DETERMINATION OF ACTIVE INGREDIENTS OF PANADOL EXTRA BY HPLC .............................. 55 COMPLEXOMETRIC TITRATIONS ................................................................................................ 58 PULVIS NEUTRACIDUS ............................................................................................................... 63 SUSPENSIO ZINCI AQUOSA ........................................................................................................ 65 ARGENTOMETRIC ANALYSIS ..................................................................................................... 66 SPARSORIUM SULFABORICUM ................................................................................................... 67 REDOX TITRATIONS .................................................................................................................. 68 SUPPOSITORIUM ANTIPYRETICUM PRO INFATE VEL PRO PARVULO ........................................... 74 INJECTIO ALGOPYRINI 50% ....................................................................................................... 76 ACIDBASE TITRATIONS ........................................................................................................... 77 SPIRITUS IODOSALICYLATUS ..................................................................................................... 80 TEST YOURSELF – SAMPLE TEST QUESTIONS ............................................................................ 82 APPENDIX .............................................................................................................................. 92 UNICAM UV/VIS SPECTROPHOTOMETER MANUAL .................................................................. 93 UV-1601 SHIMADZU SPECTROPHOTOMETER MANUAL ............................................................. 95 MARS CEM MICROWAVE DESTRUCTOR MANUAL ..................................................................... 97 ATOMIC ABSORPTION SPECTROMETER MANUAL ...................................................................... 98 HPLC MANUAL ...................................................................................................................... 100 NMR SPECTRA........................................................................................................................ 109 2
CONDUCTOMETRY
(MEASUREMENT OF SPECIFIC CONDUCTANCE)
Conductometry is based on the measurement of the conductance of electrolyte solutions.
The passage of electric current through a chemical cell is carried out by the ionic species
in the solution. It is an additive property, with the participation of all of the ions in the solution.
The conductance is specified by the measurement of the resistance of a certain segment of the
solution. The conductance (G) is the reciprocal of the resistance (R), its unit is 1/Ω Siemens;
S):
G=
1
R
The conductance is directly proprotional to the surface area (A) of the electrodes and
inversely proportional to the distance (d) between the electrodes:
1
A
=κ
R
d
κ is the specific conductance, where the resistance of the solution is measured between two
electrodes of 1 cm2 area 1 cm apart.
The conductance depends on the number of ions in the solution and on the identity of
the ions. Some ions move faster than others in an electric field, and their mobility is therefore
an important factor too.
Dilution of an electrolyte solution will decrease the specific conductance: the lower
number of ions present in a given volume, the lower the current flow is. The molar specific
conductance () was introduced to characterize of the conductance of certain ions:
= 1000

c
where c is the concentration of the electrolyte solution.
The ions in an infinitely dilute solution contribute to the conductance independently
from each other, and the molar specific conductance of an infinite dilute solution can therefore
be calculated by summing the conductances of each of the ions in the solution:

and are the conductances of cations and anions, respectively in infinitely dilute solution.
3
The electrode
Special eletrodes are used during conductometric measurements. The conductance is
determined by measurement of the resistance of the solution in a certain volume between two
electrodes made of platinized platinum. The surface area of the electrodes is increased and the
polarization resistance is decreased by platinization. The electrodes are fixed tightly in a
cylindrical unit. The fixed geometry specifies the distance of the electrodes during both the
calibration and the measurement. Alternate current is used for the conductometric analysis so
as to avoid disturbing electrode processes. A Wheatstone bridge is used to measure the
resistance.
Concentration measurement (direct conductometry) and conductometric titrations
(indirect conductometry) are distinguished in conductometry. In direct conductometry, the
concentration is determined by the measurement of conductance. This method is used, for
example, to check Aqua purificata or Aqua destillata. An electrode is built into the ion-exchange
system that continuously monitors the conductance of ion-exchanged water. When the
conductance is above a given limit, the system must be regenerated. According to the European
Pharmacopoeia 8th Edition, the maximum allowed conductance of Aqua purificata is 4.3 μS/cm,
while that of Aqua ad iniectabilia is 1.1 μS/cm.
Conductometric titrations can be applied when the ion concentration changes during a
reaction, or when the ion concentration remains constant, but the mobility of the ions changes.
Types of conductometric titrations
Acid–base titrations
It is easy to determine the equivalence point in these titrations because hydrogen ions
(H ) are the most mobile of all ions, and hydroxide ions (OH-) are the second most mobile, and
the mobilities are well above those of other ions.
+
1. Titration of a strong acid with a strong base
The titration of hydrochloric acid (HCl) with sodium hydroxide (NaOH) may serve as
an example.
4
The neutralization of HCl does not change the electrolyte concentration of the solution
before the equivalence point because the H+ are replaced by sodium ions (Na+). The lower
mobility of the Na+ results in decreased conductivity. There are two reasons why the
conductivity increases after the equivalence point. The excess NaOH increases the electrolyte
concentration in the solution, and the mobility of the OH- is high.
HCl + NaOH→H2O + NaCl
2. Titration of a weak acid with a strong base
As an example, acetic acid may be titrated with NaOH.
At the beginning of the titration, the dissociation of acetic acid is blocked by the acetate ions.
The H+ concentration is decreased, and the conductance is therefore also decreased, so that a
minimum is visible in the titration plot. The concentration of acetate ions increases on the
addition of NaOH, and the conductance increases too then slowly. Na+ also contribute to the
increase of conductance. The conductance increases sharply after the equivalence point because
of the presence of excess Na+ and OH-. The plot becomes steeper than in the previous phase
because the OH- are not neutralized and their mobility is higher than that of acetate ions.
CH3COOH + NaOH→ CH3COO- + Na+ + H2O
CH3COOH CH3COO- + H+
The intersection of the linear sections of the graph is often not clearly visible, and the
measurement is therefore not precise. This problem can be avoided by using the method
described in Section 3.
5
3. Titration of a weak acid with a weak base
As an example, oxalic acid may be titrated with N-Methylglucamine (meglumine).
Oxalic acid is a dicarboxylic acid. Its first proton (pKa1 = 1.05) can be titrated as a
medium strength acid, while the second proton (pKa2 = 4.28) can be titrated as a weak acid. Nmethylglucamine, (C6H11O5·NH·CH3), a hexosamine, is used as standard solution; it contains
a basic secondary amino group (pKa = 9.20) that can accept protons. The conductance of NMethylglucamine is negligible because its dissociation is very low.
The first proton of oxalic acid influences the conductance in the early stages of the
titration, because of its mobility. The mobile protons react with N-Methylglucamine and form
N-Methylglucammonium ions which have strongly decreased mobility. That is why the
conductance of the solution initially drops. The second proton of oxalic acid reacts according
to the following equation:
HOOC·COO- + C6H11O5·NH·CH3 → -OOC·COO- + C6H11O5·NH 2 ·CH3
The number of charged particles increases, and the conductance is therefore also
increases. The titration graph shows a decrease at the beginning until it reaches a minimum,
after which it increases slowly. After the equivalence point, N-Methylglucamine becomes
dominant in the solution. Its dissociation is practically zero and the dilution of the solution is
negligible. The conductance reaches a plateau. The intersection of the linear sections of the
graph is relatively clear, and the determination of the equivalence point is therefore easy.
6
CONDUCTOMETRIC TITRATION OF CARBOXYLIC ACIDS
Background:
Conductometric titrations are suitable for the analysis of reactions in which the ion
concentration changes or in which the concentration is kept constant but the mobility of the ions
changes.
Acetylsalicylic acid and benzoic acid are weak organic acids. N-Methylglucamine, a
weak base, is used as a standard solution for their analysis. The conductance decreases at the
beginning of the titration, and then increases moderately. The conductance does not change or
only negligibly after the equivalence point is reached. The conductance is plotted as a function
of the volume of standard solution. The intersection of the two sections is clearly visible, and
the equivalence point can be determined graphically. The conductance increases rapidly after
the equivalence point if a strong base (NaOH) is used for the analysis.
7
ANALYSIS OF ACETYLSALICYLIC ACID
Definition:
Acetylsalicylic acid contains not less than 99.5 per cent and not more than the equivalent
of 101.0 per cent of 2-(acetyloxy)benzoic acid, calculated with reference to the dried
substance.
Characters:
A white, crystalline powder or colourless crystals, slightly soluble in water, freely
soluble in ethanol. It melts at about 143°C (instantaneous method).
Quantitative analysis:
Weigh 0.1500 g acetyl salicylic acid. Prepare two independent samples! Use two 150
ml beakers. Dissolve them in 5 ml of methanol and then add 40 ml of distilled water. Put one
of your samples on the magnetic stirrer and place a stir bar in the solution. Immerse the electrode
of the conductometer into the solution and add as much water to reach the black mark on the
electrode. Turn on the conductometer with the right button under the screen. On the left of the
screen µS (micro siemens) mS (milli siemens) and °C signs are visible. The instrument shows
the actual setting that should be changed to µS range. Titrate the sample by addition of 0.5 ml
portions of 0.1 M N-methyl-glucamine (meglumine) standard solution. Record the conductance
after each 0.5 ml. Continue the titration until a total 15.0 ml of titrant is added. Plot the
conductance as a function of the volumes of the standard solution. Calculate the percentage of
acetyl salicylic acid content of the sample. Enter the result with two-decimal precision.
1 ml of standard 0.1 M N-methyl-glucamine is equivalent to 18.016 mg acetyl salicylic
acid.
Repeat the titration with 0.1 M sodium hydroxide standard solution and evaluate the
different titration curves.
1 ml of standard 0.1 M NaOH is equivalent to 18.016 mg acetyl salicylic acid.
8
ANALYSIS OF BENZOIC ACID
Definition:
Benzocaine contains not less than 99.0 per cent and not more than the equivalent of
101.0 per cent of ethyl 4-aminobenzoate, calculated with reference to the dried
substance.
Characters:
A white, crystalline powder or colourless crystals, very slightly soluble in water, freely
soluble in alcohol.
Quantitative analysis:
Weigh 0.1200 g benzoic acid. Prepare two independent samples! Use two 150 ml
beakers. Dissolve them in 5 ml of methanol and then add 40 ml of distilled water. Put one of
your samples on the magnetic stirrer and place a stir bar in the solution. Immerse the electrode
of the conductometer into the solution and add as much water to reach the black mark on the
electrode. Turn on the conductometer with the right button under the screen. On the left of the
screen µS (micro siemens) mS (milli siemens) and °C signs are visible. The instrument shows
the actual setting that should be changed to µS range. Titrate the sample by addition of 0.5 ml
portions of 0.1 M meglumine standard solution. Record the conductance after each 0.5 ml.
Continue the titration until a total 15.0 ml of titrant is added. Plot the conductance as a function
of the volumes of the standard solution. Calculate percentage of benzioc acid content of the
sample. Enter the result with two-decimal precision.
1 ml of standard.1 M meglumine is equivalent to 12.21 mg benzoic acid.
Repeat the titration with 0.1 M sodium hydroxide standard solution and evaluate the
different titration curves.
1 ml of standard 0.1 M NaOH is equivalent to 12.21 mg benzoic acid.
Notes:
The temperature of the sample is not needed to be monitored to correct the result with
a temperature factor because the temperature is kept constant during the measurement. The
instrument need not to be calibrated with solutions of known conductance as relative changes
are being recorded.
9
POTENTIOMETRIC (pH-METRIC) TITRATIONS
Visual observation of the end-point by using an acidbase indicator is simple and
convenient, but it may cause several problems. Instrumental methods are being used in most
of the quantitative analyses in the Pharmacopeia; for acidbase titrations, the measurement of
pH can be a possible solution.
Pontentiometry is an analytical method that is based on the measurement of electrode
potential. The electrode potential of the indicator electrode immersed in the analyte is used to
determine the concentration of the sample.
It is possible to measure only the potential of a cell, which is the potential difference
between two electrodes. It is universally agreed that an arbitrary electromotive force (emf) is
assigned to one electrode, and the potential of the second electrode can be measured.
Two types of galvanic cells can be distinguished:
 a cell without transmission: Ag / AgCl / ZnCl2(c1) / Zn, and
 a cell with transmission: Ag / AgCl/ KCl(c2) // ZnCl2(c1) / Zn
The major difference between the two cells is that there is an electrodeliquid interface
in a cell without transmission, while there is a liquidliquid interface in a cell with
transmission, where KCl and ZnCl2 solutions are in contact. The potential difference here is
called the liquidliquid interface potential or diffusion potential. The potential of the cells
includes the diffusion potential in every liquidliquid interface cell.
The value of the diffusion potential can be decreased by using a salt bridge. A salt
bridge consists of a concentrated or saturated solution of a specific salt, where the mobilities
of its anions and cations are nearly the same. Potassium chloride (KCl) is most frequently used
for this purpose, but when Cl- ions disturb the analysis, potassium or ammonium nitrate (KNO3
or NH4NO3) is used. A salt bridge actually means the insertion of two diffusion potentials.
As the electrolyte concentration of the salt bridge is much higher than the analyte
concentration, the two diffusion potentials are influenced by the K+ and Cl- or NH4+ and Clions. The mobilities of these ions are nearly the same, the value of the diffusion potential is
low, and the potentials at the two interfaces are also really close to each other.
The value of the diffusion potential will be very low; however, it cannot be eliminated
completely, but only decreased to a minimum.
The electrode potentials agreed by convention are determined by comparison with the
standard hydrogen electrode. The reference electrode, the standard hydrogen electrode, is set to
0.00 V. The standard hydrogen electrode is a platinized platinum electrode that is immersed in
1.0 mol/dm3 HCl and pure hydrogen gas (H2) is bubbled through it. The pressure of the H2 is
0.1 MPa. Any electrode for which the electrode potential is not yet known, can be coupled with
the standard hydrogen electrode to form a galvanic cell, and the potential of the galvanic cell
gives the the potential of the unknown electrode.
The table below shows several specific electrode potential values. The electrode
potential can be positive or negative. A negative electrode potential means that the electrode is
rather reducing relative to H+, while a positive value indicates a stronger oxidizing property
than that of hydrogen.
10
Types of electrodes in potentiometry:
1. Electrodes working on the basis of equilibrium reactions
(e.g. primary electrodes, secondary electrodes and redox electrodes)
2. Ionselective electrodes (membrane electrodes)
(e.g. pHselective glass electrodes, metal ion selective glass electrodes
and liquid membrane electrodes)
3. Moleculeselective electrodes
(e.g. enzyme electrodes and gas moleculeselective electrodes)
The pHsensitive glass electrode is the most important in the pharmaceutical analytical
practicals.
The most important part of the glass electrode that is used for pH measurement is a thin
bulbform membrane made of hydrogensensitive glass attached to a nonhydrogenselective
glass tube. The resistances of both the tube and the membrane are high. The H+ response is
given only by this special glass membrane. The depth of immersion does not influence the
measurement if the membrane is fully covered by the solution.
Table of standard electrode potentials
Li+(aq) + e- → Li(s)
-3.04
IO-(aq) + H2O(l) + 2 e- → I-(aq) + 2 OH-(aq)
0.49
K+(aq) + e- → K(s)
-2.92
Cu+(aq) + e- → Cu(s)
0.52
Ca2+(aq) + 2 e- → Ca(s)
-2.76
I2(s) + 2 e- → 2 I-(aq)
+
-
0.54
-
-
-
-
Na (aq) + e → Na(s)
-2.71
ClO2 (aq) + H2O(l) + 2 e → ClO (aq) + 2 OH (aq)
0.59
Mg2+(aq) + 2 e- → Mg(s)
-2.38
Fe3+(aq) + e- → Fe2+(aq)
0.77
3+
-
-
2+
Al (aq) + 3 e → Al(s)
-1.66
Hg2 (aq) + 2 e → 2 Hg(l)
0.80
2H2O(l) + 2 e- → H2(g) + 2 OH-(aq)
-0.83
Ag+(aq) + e- → Ag(s)
0.80
Zn2+(aq) + 2 e- → Zn(s)
-0.76
Hg2+(aq) + 2 e- → Hg(l)
3+
-
-
0.85
-
-
-
Cr (aq) + 3 e → Cr(s)
-0.74
ClO (aq) + H2O(l) + 2 e → Cl (aq) + 2 OH (aq)
0.90
Fe2+(aq) + 2 e- → Fe(s)
-0.41
2Hg2+(aq) + 2 e- → Hg22+(aq)
0.90
Cd2+(aq) + 2 e- → Cd(s)
-0.40
NO3-(aq) + 4 H+(aq) + 3 e- → NO(g) + 2 H2O(l)
0.96
2+
-
-
-
Ni (aq) + 2 e → Ni(s)
-0.23
Br2(l) + 2 e → 2 Br (aq)
Sn2+(aq) + 2 e- → Sn(s)
-0.14
O2(g) + 4 H+(aq) + 4 e- → 2 H2O(l)
2+
-
1.07
+
2-
-
1.23
3+
Pb (aq) + 2 e → Pb(s)
-0.13
Cr2O7 (aq) + 14 H (aq) + 6 e → 2 Cr (aq) + 7 H2O(l)
1.33
Fe3+(aq) + 3 e- → Fe(s)
-0.04
Cl2(g) + 2 e- → 2 Cl-(aq)
1.36
2H+(aq) + 2 e- → H2(g)
0.00
Ce4+(aq) + e- → Ce3+(aq)
4+
-
2+
+
-
1.44
-
2+
Sn (aq) + 2 e → Sn (aq)
0.15
MnO4 (aq) + 8 H (aq) + 5e → Mn (aq) + 4 H2O(l)
1.49
Cu2+(aq) + e- → Cu+(aq)
0.16
H2O2(aq) + 2 H+(aq) + 2 e- → 2 H2O(l)
1.78
ClO4-(aq) + H2O(l) + 2 e- → ClO3-(aq) + 2 OH-(aq)
0.17
Co3+(aq) + e- → Co2+(aq)
1.82
-
-
-
2-
2-
AgCl(s) + e → Ag(s) + Cl (aq)
0.22
S2O8 (aq) + 2 e → 2 SO4 (aq)
2.01
Cu2+(aq) + 2 e- → Cu(s)
0.34
O3(g) + 2 H+(aq) + 2 e- → O2(g) + H2O(l)
2.07
-
-
-
-
ClO3 (aq) + H2O(l) + 2 e → ClO2 (aq) + 2 OH (aq)
-
0.35
-
F2(g) + 2 e → 2 F (aq)
11
2.87
The inner and outer surfaces of the membrane are hydrogensensitive. The electric
potential at the outer surface, which depends on the proton concentration of the analyte, is
usually measured with a secondary electrode, e.g. Hg-Hg2Cl2 or Ag-AgCl. There is a high buffer
capacity reference solution inside the electrode where the reference electrode (usually AgAgCl) can be found.
The schematic diagram of the whole electrochemical cell:
Ag-AgCl internal Internal buffer
electrode
solution
pH sensitive
glass membrane
Analyte
solution
Outer reference
electrode
(Hg-Hg2Cl2
(Single vertical lines indicate the phase borders, while the double vertical line denotes a salt
bridge or diaphragm.)
A combined glass electrode in which the reference electrode is inbuilt is used during
the pharmaceutical analytical practicals.
Shematic diagram of a glass electrode
12
The practice of potentiometric analysis:
The measurement requires the following components:

a solution of the analyte

an indicator electrode (working (half) cell)

a reference electrode (reference (half) cell)

a potentiometer

a closed circuit (salt bridge)
Types of potentiometric analysis:

direct potentiometry

indirect potentiometry (potentiometric titration)
The concentration of the electrode active material is calculated from the emf or the
value of the electrode potential by using the Nernst-equation:
E  E0 
R T
 ln(a )
zF
where:
a = activity (a = f  c; f = activity coefficient; f  1, so a  c in the case of dilute solutions)
R = universal gas constant, 8.314 J/(mol  K)
T = absolute temperature (K)
F = Faraday constant (96,487 C/mol)
Introducing the constants:
E  E0 
0.059
log(c)
z
where
z = moles of electrons transferred in the cell reaction
c = concentration
The concentration of the electrodeactive sample can be calculated if the electrode
potential is determined (known).
Direct potentiometry is fast and easy to automatize, but there are limitations of its use
because of the possible errors.
Determination of the endpoint of a titration is also possible with potentiometric
titration. The indicator electrode is immersed in the solution of the sample that contains the
13
electrodeactive material in this case and the emf is measured as a function of the volume of
the standard solution. The titration curve is determined experimentally and its inflexion point
indicates the equivalence point of the titration. The accuracy of the measurement depends on
the determination of the endpoint of the titration and not on the accuracy of the measurement
of the emf, and thus the error will be smaller.
E  0.059  pH
The use of indicator dyes is not
necessary during the application of
potentiometry, so any indicator error is
eliminated. Potentiometric determination of
the end-point is more sensitive than visual
methods. It can be applied for solutions one
order of magnitude more dilute than those
where visual end-point determination is
used.
This method can be applied for all
the titrations where either of the reactants
can
participate
in
a
reversible
electrochemical reaction for which a
potentiometric electrode can be built. It is
used
for
neutralization
analysis,
complexometry,
argentometry
and
oxidimetry.
Determination of the equivalence point
graphically and numerically:
The precision of the determination
of the endpoint can be increased by the
derivation of the titration curve. A local
maximum or local minimum is visible in the
first derivative of the curve; while the
second derivative of the curve is zero.
The electrode potential is directly
proportional to the pH of the solution:
E  E0 
R T
 log[ H  ]
zF
14
HOW TO USE THE GLASS ELECTRODE
Combined glass electrodes are usually used when pH is monitored.
The electrode must never be allowed to dry out.
After use, the electrode must be rinsed with distilled water or, if it has been used in
some non-aqueous medium, with other appropriate solution. In special cases, washing with
water is not sufficient, and the electrode must be immersed in a cleaning solution for about
30-60 min, after which it must be rinsed and stored in an appropriate storage solution.
The electrode must be detached from the main unit before it is turned off.
The electrode must not be stored or turned upside down.
The electrode is expensive. It must be immersed into the sample solution with extreme
care, with the electrode kept about 4 cm beneath the surface. At the same time, it must be kept
as far as possible from the magnetic stirrer.
The pH-meter must be calibrated before use by immersing the electrode in different
commercially available calibrating solutions. Various calibration points may be chosen for the
calibration routine. However, calibration at two different pH values is used most frequently.
During the calibration, the electrode is attached to the main unit, and is then rinsed with distilled
water, dried and immersed in the chosen calibrating solution. The same measurement range is
set and a waiting period is necessary until the pH value on the screen has been stabilized. The
value is then recorded and stored in the memory unit of the pH-meter. After calibration, the
electrode must be rinsed again with distilled water, and the analysis can then start. The
calibration range must be chosen so that the measured pH or potentials fall into this range. The
most frequently used calibration solution is pH 7.01 buffer solution.
POTENTIOMETRIC TITRATION
The combined glass electrode is immersed into the sample solution, the magnetic
stirrer is set up for slow mixing and small quantities of standard solution are added. It is
necessary to wait for a few seconds after the addition of each volume to allow the pH to
stabilize. The pH values are recorded and plotted as a function of the volume of standard
solution used. In acidbase titrations, the temperature may rise in case of because of the
neutralizing heat. The pH may change if the temperature is not constant, and the sample flask
must therefore be thermostated.
EVALUATION OF THE MEASUREMENT
The potentiometric titration can be evaluated numerically or graphically. The
numerical evaluation may be performed manually or by computer. The pH values are plotted
as a function of the volume of standard solution used.
The inflexion points should be determined. The easiest way to find inflexion points is
the tangential method.
15
For appropriate analysis of the curves, very accurate graphs should be plotted. The
basic requirement is multiple recordings of the pH changes around the inflexion points. It is
therefore necessary to know the expected equivalence point. This is possible if the dissociation
exponents and/or the equivalence ranges are known. The standard solution is usually added in
Tangental method for evaluation of a potentiometric titration curve
to the sample 0.5-ml quantities, but in the range close to equivalence the quantities should be
smaller, e.g. 0.1-0.2 ml.
The pH change is monitored continuously, and when it changes by more then 0.1 pH
unit the added volume of standard solution should be decreased. The added volume of standard
solution can be increased again if there are two equivalence points between the two inflexion
points and the pH does not change significantly after the second inflexion point.
In order to determine the equivalence point, parallel straight lines are fitted to the
initial and final sections of the titration curve, and a third straight line is fitted to the linear
points around the inflexion points of the curve. The mean V (cm3) value of the intersections
gives the volume at the equivalence point.
Before use, the pH-meter must be set with the help of the tutor.
QUANTITATIVE ASSAYS BY TITRATION WITH ALKALINE SOLUTIONS
Titration with NaOH or KOH is used for the quantitative assay of acidic substances in
around 100 cases, in the Pharmacopoeia. More than 50% of these assays are potentiometric
titrations. Organic substances are usually dissolved in alcohol because of their limitied
solubility in water. Inorganic substances are tested in hydrophilic solutions.
16
DINATRII PHOSPHAS DIHYDRICUS
DISODIUM HYDROGENPHOSPHATE DIHYDRATE
Na2HPO4·2H2O
Mr 178.0
Definition:
Content: 98.0 per cent to 101.0 per cent (dried substance).
Characters:
Appearance: a white or almost white powder or colorless crystals.
Solubility: soluble in water, practically insoluble in ethanol (96 per cent).
Background:
Potentiometric titration is used in the Pharmacopoeia to assay the hydrated and
dehydrated forms of NaH2PO4 and KH2PO4. The samples must be dried before the analysis and
the mass loss on drying must be taken into account. The different compounds can be measured
together during the titration, which means when Na2HPO4 is analyzed, NaH2PO4 content can
also be determined. Two-step titration curves are obtained in all cases during the analysis of
these substances. An analytically accurate quantity (25.0 ml) of 1 M HCl is added at the
beginning of the measurement, and the sample is then titrated with standard 1 M NaOH.
The phosphates react with HCl to form orthophosphoric acid (H3PO4). The excess HCl
and the H3PO4 are titrated to reach the first inflexion point (V1 ml), and (25 ml - V1 ml) is
proportional to the concentration of HPO42-. The first inflexion point can be calculated by using
the dissociation exponents: ½(pKs1+pKs2); its value is ~4.6. Frequent measurement intervals
should therefore be applied around the first inflexion point at pH 4.6. The second inflexion
point is at pH ~9.7 (V2 ml), ½(pKs2+pKs3), when all the H2PO4- is converted to HPO42-. Frequent
measurement intervals should again be recorded around the inflexion point and the titration
should be continued until the pH changes is decreased dramatically (pH ~11). The result is
calculated by using the equation below.
Quantitative analysis:
Disolve 2.0000 g of sample weighed with analytical accuracy in 50 ml of water R and
add 25.0 ml of 1 M HCl. Titrate the sample potentiometrically to the first inflexion point (V1
ml) by using standard 1 M NaOH solution. Then continue the titration to the second inflexion
point (total volume of 1 M NaOH solution required V2 ml).
Calculate the percentage of Na2HPO4 by using the following formula:
1420  (25  f HCl  V1  f NaOH )
m  (100  d )
where
d = percentage loss on drying.
17
Calculate the percentage Na2HPO4 contamination of the sample according to the
following equation:
V2  f NaOH  25  f HCl
25  f HCl  V1  f NaOH
This percentage content should not be greater than 0.025%.
NATRII DIHYDROGENOPHOSPHAS DIHYDRICUS
SODIUM DIHYDROGENPHOSPHATE DIHYDRATE
NaH2PO4·2H2O
Mr 156.0
Definition:
Content: 98.0 per cent to 100.5 per cent (dried substance).
Characters:
Appearance: a white or almost white powder or colorless crystals.
Solubility: very soluble in water, very slightly soluble in ethanol (96 per cent).
Quantitative analysis
Dissolve 2.5000 g of sample weighed with analytical accuracy in 40 ml water R. Titrate
it with carbonate-free 1 M NaOH, determining the end-point potentiometrically.
1 ml of standard 1 M NaOH is equivalent to 0.120 g of NaH2PO4.
Calculate the percentage NaH2PO4 content of the powder in a similar way as for Na2HPO4.
18
CHININI HYDROCHLORIDUM
QUININE HYDROCHLORIDE
C20H25ClN2O2·2H2O
Mr 396.9
Definition:
Content: 99.0 per cent to 101.0 per cent of alkaloid monohydrochlorides,
expressed as (R)-[(2S,4S,5R)-5-ethenyl-l-azabicyclo[2.2.2]oct-2-yl]-(6-methoxyquinolin-4-yl)methanol]-hydrochloride (dried substance).
Characters:
Appearance: white or almost white or colorless, fine, silky needles, often in clusters.
Solubility: soluble in water, freely soluble in ethanol (96 per cent).
Background:
The method described below is frequently specified for the analysis of organic amine
salts amine hydrochlorides in the Pharmacopoiea. There are 78 such quantitative analyses,
including papaverine·HCl, quinine·HCl, ephedrine·HCl and pseudo-ephedrine·HCl.
The method is called displacement titration because the amine base is liberated from its
hydrochloride form during the titration. Organic amine bases are not very soluble in water, and
an alcoholic aqueous medium is therefore used. The acidic natures of the amine hydrochlorides
differ, and the sample is therefore dissolved in ethanol and before the measurement 5 ml of
0.1 M HCl is added as an adjuvant solution. This HCl is not a volumetric solution; it must be
added to the sample to allow the accurate determination of the first inflexion point, from where
the amine hydrochloride is measured. In other words, at the beginning of the titration the excess
HCl is measured by the addition of 0.1 M NaOH solution up to the first inflexion point. The
volume relating to the second inflexion point depends on the quantity of the amine base. It is
recommended to use smaller steps (e.g. 0.1-0.2 ml) around the inflexion points. If the pH jump
is more than 0.1 unit, use 0.1 ml NaOH should be used as the amount added. The quantity of
the amine base is proportional to the volume of NaOH added, which can be calculated by
subtracting the volume added up to the first inflexion point that up to the second one. There are
19
special cases when diamine dihydrochlorides are tested (e.g. histamine·2HCl,
meclozine·2HCl); in these titrations, three inflexion points can be observed.
Standardization of the 0.1 M NaOH solution
The procedure for the standardization of 0.1 M NaOH solution is similar to the
displacement titration method:
Accurately measure 0.1000 g of dried benzoic acid and dissolve it in 50 ml of alcohol.
Add 5 ml of 0.1 M HCl and titrate the solution potentiometrically with 0.1 M NaOH. 1 ml of
0.1 M NaOH is equivalent to 12.21 mg of benzoic acid. Calculate the theoretical volume by
using the equivalent mass of benzoic acid. Calculate the practical volume by subtracting the
volume added up to the the first inflexion point from the volume added up to the second
inflexion point. Calculate the factor of NaOH by using the following equation:
f 
Vtheoretical
V practical
The benzoic acid should be very pure for the standardization. If only inpure benzoic acid
is available, it should be purified in an appropriate sublimation apparatus.
Quantitative analysis:
Dissolve 0.2500 g of accurately weighed sample in 50 ml of alcohol R and add 5 ml
0.1 M HCl. Titrate the sample with 0.1 M NaOH, determining the end-point potentiometrically.
Read the volume added between the 2 inflexion points.
N.B. standard 0.1 M NaOH solution is made by the dilution of 1 M NaOH stock
solution. NaOH solutions should always be standardized because NaOH pellets are hygroscopic
and adsorb carbon dioxide (CO2). The standardization of standard NaOH solution is described
in detail in the theoretical guidelines.
1 ml of 0.1 M NaOH is equivalent to 36.09 mg of C20H25ClN2O2·2H2O.
Calculate the C20H25ClN2O2·2H2O percentage of the powder.
20
UNGUENTUM AD VULNERA
(UNG. AD VULNER.)
DERMATOLOGICUM. ANTISEPTICUM.
Composition: Acidum salicylicum
0.6 g
Vaselinum acidi borici
ad 30.0 g
Background:
At the beginning of the titration, standard 0.1 M NaOH solution is added in 0.2-ml
portions to the sample. The pH is recorded after each 0.2 ml. The pH may decrease and then
slowly increase during the titration. The change is more dramatic around the equivalence point
of the salicylic acid, after which the rate of pH increase slows down. After the equivalence
point has been reached, the sample is overtitrated by the addition of at least 5 ml of 0.1 M
NaOH solution in 0.5-ml portions and 2.0 g of mannitol is then added. Mannitol forms a
complex with boric acid (H3BO3) that can be titrated as a stronger monoprotic acid than H3BO3
itself. The pH of the solution drops by 2-3 units.
OH
OH
HO
HO
+
B
OH
HO
OH
H
NaOH
O
O
+
B
HO
O
Na + H2O
O
boric acid - mannitol complex
The titration is carried oot to reach the equivalence point of H3BO3 and continued with
5-6 ml of standard solution after the potential jump so as to be able to evaluate the result
graphically. The pH is plotted as a function of the volume of standard 0.1 M NaOH solution.
Two inflexion points are visible in the curve. The first is directly proportional to the amount
of salicylic acid. The difference between the second and first equivalence points is directly
proportional to the amount of H3BO3. The amount of salicylic acid and H3BO3 should be
calculated in 30.0 g of sample.
Quantitative analysis:
An HI 9321 type pH-meter and an HI 1331 type combined glass electrode are used
during the potentiometric measurements. The instrument can be connected to the mains through
21
an adaptor. Between measurements, the electrode is stored in a special storage solution that can
be found in the cap of the electrode. Before the measurement is started, this cap should be
removed and the electrode must be rinsed with water R before use. The lid should be kept in
such a way as to keep the storage solution intact. The electrode must be connected to the
instrument before the pH meter is turned on with the ON/OFF button. The electrode must be
immersed into the sample solution so that a distance of at least 4 cm is kept between the bottom
of the electrode and the surface of the solution. The monitor of the pH-meter shows the actual
pH (it is necessary to wait a few seconds to let the value stabilize).
Heat 1.4000 g of sample weighed with analytical accuracy with of 50.0 ml water to
100 °C, then shake it to dissolve the active ingredients, cool it down to room temperature and
filter it to remove the base of the ointment. Put the sample solution (in a 100.0 ml beaker) on a
magnetic stirrer and place a clean stir bar into the solution. Set the stirring speed to medium.
The sample solution is titrated with 0.1 M NaOH. It is recommended to use smaller quantities
at the beginning of the titration, e.g. 0.2 ml. Check and record the pH after the addition of each
quantity of NaOH (wait a few seconds after the addition of NaOH to let the pH stabilize).
During the titration, the pH first decreases slightly and increases slowly. Around the
equivalence point of salicylic acid, the pH rises rapidly, and after the equivalence point it
reaches a plateau. Continue the titration with 5-6 ml of additional 0.1 M NaOH after the
equivalence point of salicylic acid has been reached. Then add 2.0 g of mannitol to the solution.
Mannitol forms a complex with H3BO3 and this complex is a stronger acid than H3BO3 itself.
At this point, the pH of the solution drops by 2-3 units. Continue the titration with 0.1 M NaOH
to reach the next potential jump at the equivalence point of H3BO3; it is necessary to overtitrate
so that graphical determination of the equivalence points is possible.
Plot the pH values as a function of the volume of 0.1 M NaOH added. A curve with two
potential jumps gives the volumes of NaOH needed to titrate salicylic acid and H3BO3,
respectively. The volume added up to the first potential jump is equivalent to the amount of the
salicylic acid, and the difference between the second and first potential jumps is equivalent to
the amount of the H3BO3.
Calculate the salicylic acid and H3BO3 contents of a 30.0 g sample. Give the results in
grams with four-decimal precision.
1 ml of standard 0.1 M NaOH solution is equivalent to 13.812 mg of salicylic acid
(C7H6O3).
1 ml of standard 0.1 M NaOH solution is equivalent to 6.183 mg of H3BO3.
22
SPECTROPHOTOMETRY
Energy is absorbed by all atoms and compounds depending on their chemical structure.
The structure of the molecule determines the interaction of the molecule and the
electromagnetic radiation. The electromagnetic radiation absorbed is directly proportional to
the concentration of the sample, and this phenomenon can therefore be used for analytical
purposes. The method is simple, fast, sensitive, and specific, and is frequently used in
analytical chemistry for quantitative determinations.
The interpretation of absorption phenomena that occur in ultraviolet (UV) and visible
(VIS) light is at the main focus of the pharmaceutical analysis practicals. The excitation of
single σ-bonds in a molecule is very difficult; it may be achieved when far-UV light is used.
Nonbonding electrons (n-electrons) in the outer shell (that are not involved in chemical bond
formation) can be excited by UV light. π-electrons (double or triple bonds) can be excited by
both UV and VIS light.
The chromophore group of a molecule is responsible for its light absorption. Most
chromophore groups contain one or more unsaturated bonds. A group of atoms attached to a
chromophore which is able to modify how the chromophore absorbs light is called an
auxochrome. The absorption maximum of a molecule can be influenced in the following ways:
A bathochromic effect occurs when the absorption maximum shifts to longer wavelengths. The
opposite is a hypsochromic shift, when the absorption maximum shifts toward shorter
wavelengths. Hyperchromicity is the increase in absorbance of a material, while
hypochromicity is the decrease in absorbance of the substance. These phenomena are used in
practice when chromophore groups are built into a molecule:
23
Nitrobenzene is a typical chromophore; the nitro group of the aromatic ring intensifies the
conjugation. Another similar molecule is trinitrophenol (picric acid), a yellow compound; its
salts are called picrates.
The measurement of steroids in the UV-VIS range on the basis of own light absorption
at short wavelengths is really difficult. However when steroid derivatives are used on the basis
of the following reactions: the analysis can be performed:
When an aldehyde reacts with a primary amine, a Schiff base is formed. In the case of
an aromatic amine, the conjugation of the molecule is extended. A bathochromic shift occurs.
24
The determination of protein concentration is possible in the UV range at a wavelength
of 280 nm, when the absorption of the aromatic side-chains of phenylalanine, tyrosine and
tryptophan is maximal. Complex formation is often used in practice, when the protein
concentration is measured in the VIS range:
Most such measurements are based on the fact that the peptide bonds of proteins are able to
reduce copper ions (Cu2+) in alkaline media. The extent of the reaction is directly proportional
to the amount of protein in the sample. The reduced copper ions (Cu+) form a colored product
with a chelating agent, e.g. the BCA assay shown above.
25
The ninhydrin reaction is used to detect peptides/proteins. Ninhydrin can react with
primary and secondary (noncyclic) amines. The product of the reaction is a conjugated Schiff
base with an absorption maximum in VIS range. Since proline is an imino acid, it does not
react with ninhydrin.
Ninhydrin reaction
NH3 + CO2 +RCHO
O
O
OH
OH
+
H
C
R
OH
COOH
H
NH2
O
O
ninhydrin
O
O
OH
OH
+
NH3 +
H
OH
O
O
O
OH
N
O
O
blue complex, max = 570 nm
Determination of the amino groups in amino acids, peptides and proteins
The blood glucose level can normally vary between certain values. It is very important
to monitor it regularly to obtain acurate information about the carbohydrate metabolism of the
body, and particularly to identify diabetes.
Two basic techniques are used to determine the blood glucose level. The first is
chemical method, in which the nonspecific reducing property of glucose is used. The
concentration of glucose is revealed by the color and its intensity of an indicator.
26
1. Glucose + alkaline copper(II)tartrate  copper(I)-oxide
2. Cu2+ + phosphomolybdic acid  “Mo2O5” phosphomolybdic oxide (blue-colored endproduct)
The colored end-product of the reaction can be measured by spectrophotometry. The
measurement may be influenced by other reducing substances present in the blood, e.g. higher
values may be detected in uremic patients.
These problems can be eliminated by using newly developed enzymatic techniques.
The most frequently used enzymes are (a) glucose oxidase and (b) hexokinase.
(a) D-glucose + O2
glucose oxidase
D-glucono-δ-lacton + H2O2
Reducing agents, e.g ascorbic acid, bilirubin, gluthathione and certain drugs may interfere with
the determination. The method is not suitable for the determination of the blood glucose level
in urine.
(b) glucose + ATP
glucose-6-P + NADP
hexokinase
glucose-6-P-dehydrogenase
glucose-6-P + ADP
6-phosphogluconate + NADPH + H+
The reduced NADP coenzyme is determined at 340 nm.
Besides photometry, electrochemical methods, wih the use of biosensors, may be
applied to determine blood glucose levels.
Amperometric glucometers measure the current on the surface of a working electrode
due to the chemical reaction, as a function of the working electrode potential. Several factors
like temperature, hematocrit, drugs, etc., may interfere with the measurement.
The amount of substance involved in the electrode reaction can be determined by
means of Faraday’s law: the amount of substance released is proportional to the total electric
charge passed through the cell in coulometric glucometers:
27
m
M Q
nF
m = the mass of substance liberated at an electrode (g)
M = the molar mass of the substance (g/mol)
n = is the valency number of the ions of the substance (electrons transferred per ion)
F = 96,500 (C/mol), the Faraday constant
Q = the total electric charge passed through the cell (C)
The measurement is precise only if the system detects the electrons released during the redox
reaction of the analyte. The interfering reactions must therefore be blocked. Coulometric
analysis is slightly influenced by environmental factors.
The reactions on the biosensors:
D-glucose + O2
H2O2
glucose oxidase
D-glükono-δ-lactone + H2O2
platinum anode
2 H + + O 2 + 2 e-
The Beer-Lambert law describes the relationship between the concentration (c) and the
absorbance (A) of the sample; it is valid only for dilute solutions:
A  log
Io
1
 log    l  c
I
T
Transmittance is the ratio of the transmitted light and the total incident light, usually
expressed as a percentage. It is not often used in practice because its graph is hyperbolic.
T
I
Io
A = absorbance
Io = intensity of the incident light
I
= intensity of the transmitted light
T = transmittance
ε
= molar absorbance (absorbance of a 1 mol/dm3 concentration solution if l=1 cm )
l
= path length
c
= concentration of the sample (mol/dm3)
28
The analysis of a single compound in a sample is most often required. The absorption
maximum of the molecule should first be determined, after which the absorbances of the
calibration solutions are measured. The absorbance is plotted as a function of the concentration
and the concentration of the sample can then be calculated on the basis of the fitted straight line.
Multiple components in the same sample can also be analyzed. The choice of the
appropriate wavelength is critical in this case. The best choice is a wavelength where only one
of the molecules has an absorption maximum and the absorbances of the other molecules are
close to zero.
The absorbances of all the components in a sample are added:
A = Acell + Asolvent + Areagents + Aunknown sample
A’ = Acell + Asolvent + Areagents
A’ = blank solution
Aunknown sample = A – A’
The absorbance of the blank solution should be used to set the scale of the spectrophotometer
to zero because there are colored reagents such as yellow iron(III) chloride (FeCl3) solution.
Double-beam spectrophotometers are used during the practicals of pharmaceutical
analysis. A schematic diagram of the instrument is as follows:
Light
source
Monochromator
Sample
cell
Detector
Reference
cell
Double-beam spectrophotometer I.
A tungsten lamp is used as a light source in the VIS range. The lamp contains a straight
filament. The emitted electromagnetic waves cross the wall of the lamp perpendicularly. Light
sources in the UV range are the mercury-vapor lamp, the hydrogen lamp and the deuterium
lamp. The deuterium lamps used in modern spectrophotometers cover the UV range completely.
The mercury-vapor lamp is official in Ph.Hg.VIII. The disadvantage of the mercury-vapor lamp
relative to the deuterium lamp is that it has an energy minimum at ~200 nm and it cannot be
used in this wavelength region. The lamps used in spectrophotometry are filled with special
gas. Because of the high pressure inside them, the glass can cause serious injuries if they are
broken so they cannot be disposed of as communal waste. Spectrophotometers should be
turned on 10-15 minutes before use.
Prisms, half-prisms or diffraction gratings are used as monochromators in
spectrophotometers.
29
Reference
cell
Aperture
V-shaped
mirror
Light
source
Sample
cell
Detector
Beam
splitter
Double-beam spectrophotometer II.
Photocells that are especially sensitive for the electromagnetic radiation in the UV, VIS
and near IR range can be used as detectors in spectrophotometers. Incident photons excite
electrons and these free electrons fly from the cathode toward the anode when electromagnetic
waves reach the surface of the photocell. The photocurrent depends on the intensity and
wavelength of the exciting electromagnetic radiation. Semiconductors are more effective than
photocells and they operate in a broad wavelength region. CCD (charge-coupled device)
detectors are combined with photodiodes that transform light into an electronic signal. CCDs
comprise major technology in digital imaging such as in photography. In photodiode array
detectors, hundreds or thousands (e.g. 256, 512, 1024 or 2048) of photodiodes are used.
Photodiode array detectors can be used in nanometer resolution.
30
Spectroscopic methods can be distinguished by the energy of the electromagnetic radiation:
Name
Gamma-rays
X-rays
Wavelength
Effect
Practical use
0.5-10 pm
Excitation of nuclei
Material sciences, synchrotrons.
0.01-10 nm
Excitation of inner shell
electrons
Structure determination:
X-ray diffraction
Diagnostics
UV light
10-380 nm
Excitation of outer shell
electrons
Analytical chemistry: spectrophotometry.
VIS light
380-780 nm
Excitation of outer shell
electrons
Analytical chemistry: spectrophotometry.
780-2500 nm
Excitation of vibrational and
rotational states of molecules
IR radiation
2.5-300 μm
Excitation of vibrational and
rotational states of molecules
IR spectroscopy: identification tests
Microwaves
0.3 mm - 1 m
Electron spin excitation of spin
transition, excitation of
rotational states of molecules
ESR = Electron spin resonance spectroscopy
Radiowaves
1-300 m
Excitation of nuclear spins
NMR = Nuclear magnetic resonance
spectroscopy:
structure and qualitative analysis
Near-IR
31
Near-IR spectroscopy:
Quality control, identification of products
based on pigment dyes
32
PULVIS CHINACISALIS CUM VITAMINO C
(PULV. CHINACISAL. C. VIT. C)
ANTIPYRETICUM. ANALGETICUM.
Components: Chinini sulfas
0.15 g
Acidum ascorbicum
1.50 g
Acidum acetylsalicylicum
6.00 g
For 10 doses of divided powder
Background:
The acetyl group in acetylsalicylic acid can be removed by alkaline hydrolysis. The
hydrolysis is faster at higher temperature.
33
In acidic media, salicylic acid forms a violet complex with Fe3+ that can be analyzed in
the visible range.
I. Preparation of the solutions required to produce the calibration curve, and determination
of the absorption maximum of acetylsalicylic acid:
Dissolve 0.1000 g of acetylsalicylic acid R (pure reference material) weighed with
analytical accuracy in 10 ml alcohol and then add 1.25 ml of freshly prepared 10% aqueous
KOH solution. Five min later, add 0.25 ml 25% HCl to the solution and then dilute it to 100.0 ml
with water in a volumetric flask. The acetylsalicylic acid concentration of this stock solution is
1 mg/ml or 1000 µg/ml. Use this stock solution to make a 10-fold diluted solution A: mix
10.0 ml of the stock solution with 5.0 ml of 1% FeCl3 solution made with 0.1 M HCl (solution I)
and dilute it to 100.0 ml with water in a volumetric flask. The concentration of solution A is
100 µg/ml. Use solution A to prepare the solutions needed for the calibration curve. Make 20,
40, 60 and 80 µg/ml solutions in 25-ml volumetric flasks. Measure the appropriate amounts
from solution A into 25-ml flasks and make up to volume with the reference FeCl3 solution
(solution II: 5.00 ml of 1% FeCl3 made with 0.1 M HCl and diluted to 100.0 ml with water) to
keep the FeCl3 concentration constant (0.05%). The 100 µg/ml solution is solution A itself. (If
the weight of salicylic acid is different from 0.1000 g, the concentrations will be different, and
that must be taken into account during the calculation.)
Amount of solution A to prepare:
Absorbance
20 µg/ml solution
ml
40 µg/ml solution
ml
60 µg/ml solution
ml
80 µg/ml solution
ml
Determine the absorption maximum of acetylsalicylic acid according to the manual of
the spectrophotometer between wavelengths of 500 and 600 nm. (The 60 µg/ml calibration
solution should be used.) Measure the absorbances of the calibration solutions at the absorption
maximum of the acetylsalicylic acid. The spectrophotometer draws the calibration curve; check
and record the r2 value.
Preparation of the sample for determination of its acetylsalicylic acid concentration:
Dissolve 80 mg of substance in 10.0 ml of alcohol then add 1.25 ml of freshly prepared
10% KOH solution. Five min later, add 0.25 ml of 25% HCl and then dilute it up to 100.0 ml
with water. Mix 10.0 ml of this solution with 5.0 ml of 1% FeCl3 solution made with 0.1 M
HCl (solution I) and dilute to 100.0 ml with water.
Calculate the acetylsalicylic acid content of the powder, using the concentration
calculated by the spectrophotometer on the basis of the calibration curve.
Determine the specific absorbance of the acetylsalicylic acid too.
34
Background:
The concentrations of multiple components in a sample can be analyzed by spectrophotometry. Measurements can be made when a wavelength can be found where one compound
has an absorption maximum and the absorbances of the other compounds are zero. Appropriate
wavelengths can be found for two-component samples, but the probability decreases when
three- or multiple-component samples are to be analyzed. The amounts of both acetylsalicylic
acid and quinine sulfate can be determined by spectrophotometry in Pulivis chinacisalis.
Acetylsalicylic acid absorbs UV light, but its absorption maximum is shifted toward the VIS
range (bathochromic effect) as a result of complex formation, and therefore quinine sulfate does
not influence the measurement.
The absorbance changes rapidly if a peak is sharp, whereas the change is not so dramatic
if peak is broad. The shape of the absorption maximum peak should be considered during the
measurement. Choosing a broad peak is more appropriate, as indicated in the figure above. The
measurement wavelength chosen for the analysis is closer to the absorption maximum in the
case of a broad peak.
II. Preparation of the solutions required to produce the calibration curve, and determination
of the absorption maximum of quinine sulfate:
Dissolve 50.0 mg of accurately pure quinine sulfate R weighed with analytical accuracy
with gentle heating in a mixture of 1.0 ml 0.05 M H2SO4 and 10.0 ml of alcohol. Cool the
solution to room temperature and dilute it to 50.0 ml with water. The quinine sulfate
concentration of this solution is 1.0 mg/ml. Prepare a 10-fold dilution in a 50.00-ml volumeric
flask with the solution that contains 2.50 ml of 0.05 M H2SO4 and 25.0 ml of alcohol per 250.00
ml. The concentration of the 10-fold diluted solution is 100 µg/ml. (If the weight of quinine
sulfate is different, the concentration will be different and that must be taken into account.) Use
35
the 100 µg/ml solution to make the following calibration solutions in 25.0-ml volumeric flasks:
10.0, 20.0, 30.0, 40.0 and 50.0 µg/ml. Use the above-mentioned acidic alcoholic solution to
make the dilutions.
Amount of 100 µg/ml solution to prepare:
Absorbance
10 µg/ml solution
ml
20 µg/ml solution
ml
30 µg/ml solution
ml
40 µg/ml solution
ml
50 µg/ml solution
ml
Use the 30 µg/ml calibration solution to determine the absorption maximum of quinine
sulfate between wavelengths of 300 and 400 nm according to the manual of the
spectrophotometer. When the absorption maxima are known, set the spectrophotometer to the
longer wavelength and determine the absorbances of the calibration solutions. The reference
solution is the acidic alcoholic solution used for the dilutions.
The spectrophotometer draws the calibration curve; check and record the r2 value.
Preparation of the sample for determination of its quinine sulfate concentration:
Dissolve 0.1500 g of sample weighed with analytical accuracy with gentle heating in
the mixture of 1.0 ml of 0.05 M of H2SO4 and 10.0 ml of alcohol. Cool the solution to room
temperature and dilute it to 100.0 ml with water. Measure the absorbance of the sample.
Calculate the quinine sulfate content of the powder, using the concentration calculated
by the spectrophotometer on the basis of the calibration curve.
Determine the specific absorbance of the quinine sulfate too.
N.B. The powder is made only for the pharmaceutical analysis practicals. The acetylsalicylic
acid and quinine sulfate contents may be different from those in the original formulation.
36
TABLETTA ASPIRINI 500
(ASPIRIN TABLET 500)
ANTIPYRETICUM. ANALGETICUM.
Composition: Acidum acetilsalycilicum
500 mg
Cellulosum (pulvis)
qu. s.
Amylum maydis
qu. s.
for each tablet
Preparation of the solutions required to produce the calibration curve, and determination of
the absorption maximum of acetylsalicylic acid:
Dissolve 0.1000 g of acetylsalicylic acid R weighed with analytical accuracy in 10 ml
of alcohol and add 1.25 ml of freshly prepared 10% KOH. Five min later, add 0.25 ml 25% HCl
to the solution and then dilute it to 100.0 ml with water in a volumetric flask. The acetylsalicylic
acid concentration of this stock solution is 1 mg/ml or 1000 µg/ml. Use this stock solution to
make a 10-fold diluted solution A: mix 10.0 ml of the stock solution with 5.0 ml of 1% FeCl3
made with 0.1 M HCl (solution I) and dilute it to 100.0 ml with water in a volumetric flask. The
concentration of solution A is 100 µg/ml. Use solution A to prepare the solutions of the
calibration curve. Make 20, 40, 60 and 80 µg/ml solutions in 25-ml volumetric flasks. Measure
the appropriate amounts from solution A into 25-ml flasks and make them up to volume with
the reference FeCl3 solution (solution II: 5.0 ml of 1% FeCl3 made with 0.1 M HCl and diluted
to 100.0 ml with water) to keep the FeCl3 concentration constant (0.05%). The 100 µg/ml
solution will be solution A itself. (If the weight of the salicylic acid differs from 0.1000 g, the
concentrations will differ and this must be taken into account during the calculation.)
Amount of solution A to prepare: Absorbance
20 µg/ml solution
ml
40 µg/ml solution
ml
60 µg/ml solution
ml
80 µg/ml solution
ml
Determine the absorption maximum of acetylsalicylic acid between 500 and 600 nm
according to the manual of the spectrophotometer. (The 60 µg/ml calibration solution should
be used.) Measure the absorbances of the calibration solutions at the absorption maximum of
the acetylsalicylic acid.
The spectrophotometer draws the calibration curve; check and record the r2 value.
Preparation of the sample for determination of its acetylsalicylic acid concentration:
Weigh an intact pill with analytical accuracy into a 100-150-ml beaker and add 20.0 ml
of alcohol, and then add 10.0 ml freshly prepared 10% KOH solution. Cover the beaker with a
watch glass and boil the sample for 5 min. Cool the sample to room temperature, add 1.0 ml of
25% HCl and dilute make the volume up to a 100.0 ml with water in a volumetric flask. Mix
37
1.00 ml of this solution with 5.0 ml of 1% FeCl3 made with 0.1 M HCl (solution I) and dilute
it to 100.0 ml with water.
Calculate the acetylsalicylic acid content of the powder, using the concentration
calculated by the spectrophotometer on the basis of the calibration curve.
Determine the specific absorbance of acetylsalicylic acid too.
38
SUPPOSITORIUM PARACETAMOLI 500 MG
(SUPP. PARACET. 500 MG)
ANTIPYRETICUM. ANALGETICUM.
Composition: Paracetamolum
3.00 g
Adeps solidus 50 : Butyrum cacao
7:3
qu. s.
for 6 suppositories
Background:
The following factss should be borne in mind when measurements are made in the UV
range.
Solvents that absorb UV light cannot be used, e.g. benzene or toluene.
Some solvents can absorb UV light at shorter wavelengths (e.g. ~180 nm), which
disturbs the analysis. In measurements at 200-210 nm, the absorption is independent of the
structure of the the molecule. The absorption is slightly dependent on the structure at ~250 nm,
and therefore it is very important to note
the absorption maxima. The absorption peak is definitely not specific for the analyte when the
absorption maximum is at ~200 nm and the absorbance is higher than 1 (A>1). The
measurement should be performed by setting the spectrophotometer to the absorption maximum
of the second peak, e.g. at 280 nm in the figure above.
39
Preparation of the solutions required to produce the calibration curve, and determination of
the absorption maximum of paracetamol:
Weigh 0.0500 g of paracetamol R with analytical accuracy, dissolve it in 5 ml of
chloroform and add methanol to make it up to volume in a 50-ml volumetric flask (solution A).
Dilute 1 ml of solution A with methanol to 50 ml (solution B) and use this solution to make 2.0,
4.0, 6.0, 8.0 and 10.0 µg/ml calibration solutions in 25-ml volumeric flasks.
Amount of solution B to prepare
Absorbance
2 µg/ml solution
ml
4 µg/ml solution
ml
6 µg/ml solution
ml
8 µg/ml solution
ml
10 µg/ml solution
ml
Determine the absorption maximum of paracetamol by using the 6.0 µg/ml calibration
solution between 200 and 300 nm according to the manual of the spectrophotometer. When the
absorption maxima are known, set the spectrophotometer to the longer wavelength and
determine the absorbances of the calibration solutions. The reference solution is the acidic
alcoholic solution used for the dilutions.
The spectrophotometer draws the calibration curve; check and record the r2 value.
Preparation of the sample for determination of its paracetamol concentration:
Weigh 0.3600 g of suppository, which contains ~60 mg of paracetamol, melt it on a hot
plate and mix it with 10.0 ml of chloroform. Add methanol to make it up to volume in a
100-ml volumetric flask. Wait 20-30 min and then dilute 0.5 ml of the clear supernatant up to
50.0 ml with methanol. Measure the sample solution at the same wavelength and determine its
concentration by using the calibration curve.
Calculate the paracetamol content of a suppository of average weight.
Calculate the specific absorbance of paracetamol.
40
SPARSORIUM ANTISUDORICUM
(SPARS. ANTISUDOR.)
DERMATOLOGICUM. ANTISUDORICUM. ADSTRINGENS. DESODORANS.
Composition: Hexachlorophenum
0.60 g
Acidum salicylicum
1.80 g
Alumen
6.00 g
Magnesii subcarbonas
20.00 g
Zinci oxydum
20.00 g
Talcum
20.00 g
Background:
Preparation of the solutions required to produce the calibration curve, and determination of
the absorption maximum of salicylic acid:
Weigh 0.0500 g of salicylic acid R with analytical accuracy into a small beaker and
dissolve it in a few ml of methanol and then wash the solution into a 50.0-ml volumetric flask
with methanol and make it up to volume with the same solvent. Dilute 5 ml of the previous
solution with solution II (5.00 ml of FeCl3 solution made with 1% 0.1 M HCl and come up to
volume 100.0 ml with water) in another 50.0-ml volumeric flask. The salicylic acid
concentration of this solution is 100 µg/ml. (If the weight of the salicylic acid is different, the
concentration will be different.) Use this solution to prepare 10, 20, 30, 40 and 50 µg/ml
calibration solutions in 25.0-ml volumetric flasks, using the same solution II for the dilutions.
41
Amount of 100 µg/ml solution to prepare
Absorbance
10 µg/ml solution
ml
20 µg/ml solution
ml
30 µg/ml solution
ml
40 µg/ml solution
ml
50 µg/ml solution
ml
During the spectrophotometric measurements, use solution II as reference solution. Use
the 30 µg/ml calibration solution to record the absorption curve according to the manual of the
spectrophotometer and determine the absorption maximum of the curve. Measure the
absorbances of the calibration solutions
The sSpectrophotometer draws the calibration curve; check and record the r2 value.
Preparation of the sample for determination of its salicylic acid concentration:
Weigh 0.60 g of material, mix it vigorously with methanol and make the final volume
up to 50.0 ml with the same solvent. Filter the suspension, throw away the first 10 ml and
continue the filtration through the same filter paper into a clean beaker. Take 5 ml of the second
filtrate to make a 10-fold diluted solution, using solution II (5.00 ml FeCl3 made with 1% 0.1 M
HCl per and come up to volume 100.0 ml with water). Measure the sample solution an the same
wavelength and determine its concentration by using the calibration curve.
Calculate the salicylic acid content of the sample.
Determine the specific absorbance of salicylic acid.
42
SOLUTIO METRONIDAZOLI
(SOL. METRONIDAZ.)
ANTIAPHTOSUM.
Composition: Metronidazoleum
0.30 g
Lidocainum
0.05 g
Glycerinum
20.00 g
Ethanolum 70%
ad 30.00 g
Preparation of the solutions of the calibration curve, determination of the absorption
maximum of metronidazole:
Weigh 0.0500 g of metronidazole R into a small beaker, dissolve it in methanol and
wash the solution into a 50.0-ml volumeric flask (solution A). Prepare a 10-fold diluted
solution: transfer 5.0 ml of solution A into a 50-ml volumetric flask and make it up to volume
with methanol (solution B). The concentration of solution B is 0.100 mg/ml (100 µg/ml). (If
the weight of the metronidazole differs from 0.0500 g, the concentration of the solution will
differ and this should be taken into account.) Use solution B to make 2.0, 5.0, 10.0, 15.0 and
20.0 µg/ml calibrating solutions in 25-ml volumeric flasks.
Amount of solution B to prepare
Absorbance
2 µg/ml solution
ml
5 µg/ml solution
ml
10 µg/ml solution
ml
15 µg/ml solution
ml
20 µg/ml solution
ml
Determine the absorption maximum of metronidazole using the 10.0 µg/ml calibration
solution, scanning the region between 300 and 400 nm according to the manual of the
spectrophotometer. Measure the absorbances of the calibration solutions at the absorption
maximum of metronidazole.
The spectrophotometer draws the calibration curve; check and record the r2 value.
Preparation of the sample for determination of its metronidazole concentration:
Weigh 0.5000 g of sample and dilute it to 50.0 ml with methanol. Transfer 5.00 ml of
the solution to a 50.0-ml volumetric flask and make it up to volume with methanol. Measure
the absorbance of the sample solution at the same wavelength and determine its concentration
by using the calibration curve.
Calculate the metronidazole content of the sample.
Determine the specific absorbance of metronidazole.
43
PULVIS CHOLAGOGUS
(PULV. CHOLAGOG.)
CHOLAGOGUM. SPASMOLYTICUM.
Composition: Homatropini methylbromidum
0.03 g
Phenolphthaleinum
0.50 g
Papaverini hydrochloridum
0.60 g
Acidum dehydrocholicum
2.50 g
Natrii salicylas
2.50 g
Natrii bensoas
2.50 g
For 10 doses of powder
Background:
Phenolphthalein turns colorless in acidic solutions and is pink in basic solutions. If the
concentration of the indicator is particularly strong, it can appear purple. In very strongly basic
solutions, the pink color of phenolphthalein fades and it becomes colorless again.
Phenolphthalein is often used as an indicator in acidbase titrations. Its earlier use as a as
laxative was stopped recently because its long-term application can cause malignancy.
Phenolphthalein absorbs UV light in both its protonated and its deprotonated forms, due to its
three aromatic rings. In the deprotonated form (slightly alkaline pH), the delocalization extends
to the entire molecule. The excitation energy is decreased by the more extensive delocalization,
and longer wavelengths (VIS) can therefore be used for its quantitative analysis.
Phenolphthalein absorbs green light and transmits the complementary color pink (the one we
see).
The pH of the sample is shifted into the alkaline range by Na2CO3 during the analysis of
phenolphthalein.
44
Barbiturates exhibit a similar phenomenon. The heterocyclic system of barbituric acid
can be is stabilized in the oxo form at acidic pH, while the enol form is stable at alkaline pH.
Barbituric
acid shows both enol-oxo and lactam-lactim tautomerism. Barbiturates (5,5-disubstituted
derivatives) shows only lactam-lactim tautomerism.
Buffering of the sample solutions is therefore very important. However, possible decomposition
reactions, e.g. acidic or alkalic hydrolysis must be avoided.
The absorption spectrum changes as a function of pH. The molecule has different
absorption maxima and minima at different pH values. The wavelength at which the absorbance
is independent of the pH is called the isobestic point. This fact should be remembered during
measurements in clinical chemistry. If the pH is set wrongly, the result will be false. Isobestic
points of reference materials can be used to calibrate the spectrophotometer.
Preparation of the solutions required to produce the calibration curve, and determination of
the absorption maximum of phenolphthalein:
Weigh 0.0060 g of phenolphthalein R and dissolve it in methanol using a 100-ml
volumetric flask (solution A). Take 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml, and 5.0 ml portions of
solution A and dilute them with 0.1 M Na2CO3 to 50.0 ml.
45
Concentration of the calibration
solution
Amount of solution A
µg/ml solution
1 ml
µg/ml solution
2 ml
µg/ml solution
3 ml
µg/ml solution
4 ml
µg/ml solution
5 ml
Absorbance
The reference solution is 0.1 M Na2CO3. Determine the absorption maximum of
phenolphthalein by using the 3rd calibration solution, scanning the region between 500 and
700 nm according to the manual of the spectrophotometer. Measure the absorbances of the
calibration solutions at the absorption maximum of phenolphthalein.
The spectrophotometer draws the calibration curve; check and record the r2 value.
Preparation of the sample for determination of its phenolphthalein concentration:
Weigh 0.0300 g of sample and dissolve it in methanol in a 100.0-ml volumetric flask.
Prepare two dilutions: take 4.0 ml and 8.0 ml in different 50.0-ml volumetric flasks and dilute
them with 0.1 M Na2CO3. Measure the absorbances of the sample solutions at the same
wavelength and determine their concentrations by using the calibration curve.
Calculate the mass percent phenolphthalein content of the sample with four-decimal
accuracy.
Determine the specific absorbance of phenolphthalein.
46
DETERMINATION OF PROTEIN CONCENTRATION WITH THE BIURET REAGENT
The proteins are macromolecules that have a large variety of functions and are essential
for human life. They are built up from 20 amino acids, which are connected through peptide
(amide) bonds. 50% of the dry material of a cell is protein. The total protein concentration in
the human body is ~60-80 g/l.
Background:
Several methods are known for the determination of protein concentration. Some
methods make use of the properties of peptide bonds or an amino acid side chains that react
with certain reagents or dyes forming colored complexes. One well-known method is called
the biuret reaction. The reagent, which contains Cu2+ ions in alkaline medium, forms bluishviolet complexes with peptide bonds. At least two peptide bonds are necessary for the reaction.
Proteins fulfil this requirement in all cases. The following complex is formed:
The biuret reagent contains CuSO4, NaOH, K,Na-tartrate and KI. (The K,Na-tartrate is
necessary to keep Cu2+ in solution in the alkaline medium. KI stabilizes the reagent, so that the
shelf life becomes longer.)
Preparation of the solutions required to produce the calibration curve, and determination of
the absorption maximum of BSA:
20 mg/ml of protein stock solution made from BSA (bovine serum albumin) is used for
the calibration solutions. Pipette 700 μl of biuret reagent into each of the test tubes and then add
the following components according to the table:
47
Number of test
tube
Dist. water
(ml)
BSA stock
solution
(ml)
1
0.3
0
2
0.3
0
3
0.275
0.025
4
0.250
0.05
5
0.225
0.075
6
0.200
0.1
7
0.175
0.125
Concentration
(mg/ml)
Absorbance
Incubate the reaction mixture tubes in a 35-40 C water bath for 10 min.
Calculate the concentrations of the calibration solutions (column 4). 10 min later, use
tube 5 to determine the absorption maximum in the range 500-700 nm. The reference solutions
are in tube 1 and tube 2, which do not contain BSA. Measure the absorbances of the calibration
solutions at the absorption maximum of BSA.
The spectrophotometer draws the calibration curve; check and record the r2 value.
Preparation of the sample and determination of its protein concentration:
Take aliquots of 75 μl-t and 125 μl from the unknown sample into separate test tubes.
Dilute them up to 300 μl with distilled water and then add 700 μl of biuret reagent. The
measurement is more precise if the unknown sample is washed into a mixture of distilled water
and the biuret reagent. It is recommended to pipette the reagent first, then add distilled water
and finally wash the required amount of sample into the reaction mixture.
Measure the absorbances of the samples.
Calculate the protein concentration and give the final result in g/l with two-decimal
accuray. Average the results on the individual samples.
48
ATOMIC ABSORPTION SPECTROMETRY
Methods of sample preparation
The following methods are used in the pharmaceutical industry to prepare samples for
atomic absorption spectrometric measurements:

dry ashing

wet decomposition
o in open vessels at atmospheric pressure
o in closed Teflon beakers with steel covers, which allows the use of high
pressure
o with microwave radiation in closed plastic vessels: microwave-assisted
sample preparation
Microwave-assisted sample preparation will be described in detail here as this
technique will be used during the practicals.
The microwave-assisted wet decomposition of samples is becoming widely used in the
pharmaceutical industry. This technique is suitable for the preparation of both solid and liquid
samples. The procedure is carried out in a microwave oven. The inner wall of the oven is made
of acid-proof steel covered with Teflon. The samples are fixed on a rotating plate in the
microwave oven. These instruments have built-in pressure control units; the latest ones can
also monitor the temperature. The sample vessels are made of acid-, temperature- and pressureproof plastic.
The samples are weighed directly into the plastic vessels. 2–3 ml of liquid or 0.2–0.5 g
solid sample is usually weighed for decomposition and concentrated H2SO4, HCl, HNO3,
and/or H2O2 is then added. The membrane in the pressure valve should be checked. The vessels
are then thightly sealed with the appropriate tool (torque wrench) and placed into a Kevlar
jacket. The pressure valve opens only if overpressure develops. If the membranes are damaged,
acid vapor is released and the samples are not suitable for pharmaceutical analysis, but the
vessels remain unaffected. After the microwave treatment, the samples should be cooled to
room temperature. During the process the carbon and hydrogen present in the organic
components of the samples are turned into water and CO2 by the end of the wet decomposition;
the inorganic compounds are dissolved in water or in the applied acids. The cooled samples
are transferred into volumetric flasks for the analytical measurements, the vessels must also be
rinsed out. Water molecules and other dipolar solvents are affected by the microwave
treatment, making them oscillate with high frequency. This phenomenon heats the samples
rapidly. The advantages of the microwave-assisted wet decomposition are:

only small amount of reagents are necessary

the closed system keeps the corrosive vapors inside the vessel

only a short time is required for sample preparation
Atomic spectroscopy
Each element has a unique atomic electron configuration, and therefore a characteristic
atomic spectrum. As each spectral line is sharp, the spectral lines of different elements do not
or only rarely overlap.
49
Most elements vaporize at elevated temperatures and the molecules then dissociate into
free atoms. The first step in atomic spectroscopy is the conversion of molecules into free atoms
in the gaseous phase. If the atoms are then converted into an excited phase, they emit
characteristic radiation when they relax. Analysis of the emitted radiation provides analytical
information. These are the basic principles of the atomic spectroscopy. In other cases, the
vaporization and atomization are not accompanied by excitation of the atoms, and the basis of
the analytical measurements is the absorption of electromagnetic radiation by specific light
sources. This latter technique is atomic absorption spectroscopy (AAS).
Both methods are very effective, with detection limits in the ppm (parts per million)
range, and the inductively coupled plasma (ICP) method (see below) is even suitable for the
analysis of ppb (parts per billion) concentrations. As these are sensitive methods, problems
arise from the aspect of accuracy. The methods are less accurate than, for instance,
spectrophotometry.
Atomic absorption spectroscopy
AAS is useful for the analysis of more than 70 metals and semimetallic elements. Only
a small amount of sample is usually used for the analysis.
In the atomization unit of the instrument, the electrons of the atoms are promoted to
higher orbitals by absorbing definite quantities of energy (e.g. in the cases of certain
electromagnetic waves). The energy required for the excitation is specific for the electron
transition of the element; a unique wavelength is specific for a certain element. The method is
therefore selective.
The sample is transformed into the gaseous phase and atomized during the analysis.
Light passed through the atomized sample and is absorbed by the sample. The rate of
absorption is directly proportional to the concentration of the analyte. Before the analysis,
calibration solutions with known concentration must be prepared and the instrument must be
calibrated.
The major units of the instrument are the light source, the sample introduction unit, the
atomization unit, the monochromator and the detector. The various spectrometers may differ
in the atomization unit and the sample introduction unit. The methods available for sample
analysis are flame, electrothermal and cold vapor-hydride atomization.
Hollow cathode lamps are most commonly used as light sources in AAS. High voltage
(100-400 V) is applied across the anode and cathode, resulting in ionization of the gas inside
the sealed lamp. The emitted radiation is characteristic of this fill gas and the material of the
cathode. The fill gas is usually Ar or Ne, depending on the analyte element. The spectral lines
of the fill gas should not overlap with the analyte element. The cathode is made of the analyte
element or covered with it. The electrons are accelerated between the cathode and the anode
and ionize the fill gas, and the gas ions are therefore accelerated toward the cathode, causing
the sputtering of atoms from the cathode. The atoms of the cathode become excited upon
collisions with the fill gas and emit light as they fall back to the ground state. The emitted light
is characteristic of the element. The intensity of the hollow cathode lamp can be influenced by
the current.
The atomization unit produces free atoms capable of the absorption of electromagnetic
radiation. The atomization is either chemical or thermal; the latter is used for all elements
except Hg. Three methods are available for thermal atomization: flame, electrothermal furnace
(graphite tube) or radiofrequency-induced plasma. The sample is sprayed through the nebulizer
into the flame, where the solvent is first evaporated rapidly. The molecules are atomized due
50
to the high temperature. A large amount of sample is usually required to obtain a continuos
signal during the measurement.
Premixed laminal gases are used in flame atomization. This type of flame is stable, the
background is low and the transformations of the sample are separated throughout the height
of the flame. Two types of gas mixes are used in most cases: acetyleneair and acetyleneN2O.
Their temperatures differ; one is ~2000 K and is capable of atomizing most elements. The
analytes should be volatile, e.g. Cl-, F- or H- salts. Other elements may form stable oxides at
high temperatures, (e.g. Al, Ti, V, and W) and higher temperatures are necessary for their
atomization; this is provided by acetyleneN2O (~3000 K). The atomization may be blocked
by oxygen either in the flame or in the sample, resulting in the formation of oxides that
dissociate only at very high temperatures. A reducing flame is therefore used, when the amount
of fuel gas is higher than the oxidant gas.
Flame atomization requires a large amount of sample, whereas in the case of a graphite
furnace several µl of sample is sufficient and solid samples can also be analysed. The graphite
furnace is heated up gradually to the appropriate temperature. The sample is first dried at
~400 K, and is next heated to 1000-1500 K to ash all the organic molecules. Finally, at 3000 K,
the sample evaporates and atomizes. The gradual heating is necessary to avoid smoke in the
light path. The graphite furnace serves as a cell. Graphite is an extremely good reducing agent
at 2800-3200 K and therefore blocks the formation of metal oxides. The efficiency of the
atomization is improved too. The sensitivity of this technique is 2–3 orders of magnitude higher
than that of flame atomization. The method is sutable for the analysis of volatile samples.
The emission spectra of hollow cathode lamps consist of multiple spectral lines due to
the elements present: the material of the cathode, the fill gas (He or Ne) and the contaminants,
if any. A monochromator system is therefore necessary for the selection of the appropriate
wavelength. Littrow monochromators are usually used for this purpose. A spectral width of
0.2 nm is suitable for the AAS measurements. Narrowing the width of the slit is necessary until
the spectral lines are well separated; there is no advantage of further narrowing.
Photomultipliers are used for the detection of the intensity of the transmitted light. The
current produced by the incident light is multiplied approximately 108-fold. The sensitivity and
the stability of the photomultiplier are critical. The sensitivity depends on the photocathode
material, while the stability is affected by the high-voltage electronic system. The magnitude
of the high voltage influences the output of the photomultiplier.
To be able to analyze multiple elements in the same sample, it was necessary to
improve not only the light source, but also the detection methods. The photomultiplier was
changed to a photodiode, a photodiode array, a CCD (charge-coupled device), or a CID
(charge-injection device), so that simultaneous detection at multiple wavelengths became
possible, the detection of inhomogeneity was solved, and the errors caused by the
inhomogeneous dispersion of atoms were eliminated.
Metal atoms absorb characteristic electromagnetic radiation, depending on their
concentration. The detector measures the intensity of the transmitted light. The relationship
between the absorbance and the concentration of the analyte follows the Beer-Lambert law:
A  lg
Io
  l c
I
or A    c
51
A number of elements in the same sample can be analyzed by AAS. The method is
sensitive; it is capable of detecting as low as ppm concentrations, and is suitable for trace
element analysis.
ICP atom emission spectrophotometry
The torch of the ICP (inductively coupled plasma) is heated to extremely high
temperatures, and it is suitable for emission spectroscopic measurements as the samples are
converted into an excited state in that temperature range.
The ICP light source consists of three concentric quartz tubes in which different gases
flow. The outer gas serves as a cooling gas, preventing the external quartz chamber from
melting because of the high temperature (10,000 K). The quartz tubes are transparent and do
not absorb the electromagnetic waves used during the analysis. The gas in the middle chamber
elevates the plasma, while the gas in the middle of the light source delivers the sample into the
plasma. Ar is usually used during the analysis. A coil of the radiofrequency (RF) generator
surrounds part of this quartz torch. The Ar gas is ionized after the ignition of the torch. The RF
generator creates an intense electromagnetic field that forces the charged particles to accelerate.
A stable, high-temperature plasma of about 6,000-10,000 K is then generated as a result of the
collisions between the neutral Ar atoms and the charged particles.
The introduction of the samples depends on their state, their quantity, their physicochemical properties and the concentrations of the analytes. The solutions are simply sprayed
by the nebulizer into the flame. Pneumatic, ultrasonic and high-pressure hydraulic nebulizers
are usually used. V-shaped or Babington nebulizers are widely used, which are suitable for
transferring solutions or suspensions without the risk of clogging capillaries 0.5-1.5 mm in
diameter.
The accuracy of the measurement is influenced by changes in the amount of charged
particles during the analysis.
The detection limits of the recently used instruments are below ppb for many elements,
and they are therefore suitable for the analysis of biological samples containing low
concentrations of metal ions.
Uses of AAS and emission spectroscopy in the pharmaceutical industry:

for the analysis of multivitamin complexes

for the analysis of blood/serum, e.g. determination of Cu, As and Se

for the analysis of medicines, e.g. the presence and amounts of reduction
catalysts

for the determination of contaminants

for environmental analysis, e.g. Pb content determination
52
DETERMINATION OF MAGNESIUM CONTENT OF SPARSORIUM ANTISUDORICUM
BY FLAME ATOMIC ABSORPTION
Composition: Hexachlorophenum
0.60 g
Acidum salicylicum
1.80 g
Alumen
6.00 g
Magnesii subcarbonas
20.00 g
Zinci oxydum
20.00 g
Talcum
20.00 g
Preparation of the sample:
Weigh 0.5000 g of sample into the container of the microwave destructor. Use a clean
weighing boat. Add 20 ml of 4% H2SO4 to the sample. Wash the material off the wall. Set the
container of the microwave destructor. Use the GYAK-HP500 menu to treat the sample. N.B.
Be careful during the opening of the container after the microwave treatment because the
sample might be very hot. Cool the sample and filter it. Wash the filtrate into a 100-ml
volumetric flask and make it up to volume with water. Transfer 0.5 ml of the solution into a
50.0-ml volumetric flask and make it up to volume with 4% H2SO4 (solution A). Transfer
portions of 5.0 ml and 10.0 ml from solution A into 100.0-ml volumetric flasks and make them
up to volume with 4% H2SO4.
Determination of the calibration curve and the magnesium concentration of the sample:
Prepare calibration solutions with 0.20, 0.30, 0.40, 0.60 mg/l Mg concentration from
100 mg/l Mg solution. Transfer the aliquots into 25.0-ml volumetric flasks and make up the
volume with 4% H2SO4.
100 mg/l Mg solution
0.20 mg/l
0.30 mg/l
0.40 mg/l
0.60 mg/l
Measure the absorbances of the calibration solutions at 285.2 nm and 0.7 nm bandwidth
and determine the calibration curve.
Calculate the Mg content of the powder.
53
DETERMINTION OF MAGNESIUM CONTENT OF PULVIS NEUTRACIDUS BY
FLAME ATOMIC ABSORPTION
Composition: Calami rhizoma
1.0 g
Frangulae cortex
1.0 g
Natrii hydrogenocarbonas
8.0 g
Bismuthi subnitras
14.0 g
Magnesii subcarbonas
16.0 g
For 40 divided powders
Preparation of the sample:
Weigh 0.2500 g of sample into the container of the microwave destructor. Use a clean
weighing boat. Add 10 ml of 4M HNO3 to the sample. Wash the material off the wall. Set the
container of the microwave destructor and place it into the instrument. Use the GYAK-HP500
menu to treat the sample. N.B. Be careful during the opening of the container after the
microwave treatment because the sample might be very hot. Wash the sample into a 100.0-ml
volumetric flask and make up the volume with water.
Determination of the calibration curve and the magnesium concentration of the sample:
Prepare a 500-fold diluted solution from the Mg stock solution in a 50.0-ml volumeric
flask (solution A). Prepare the calibration solutions in 25.0-ml volumeric flasks from solution
A. Make them up to the volume with 0.2 M HNO3. Prepare calribration solutions with Mg
concentrations 0.25, 0.50, 0.8 and 1.00 mg/l.
Amount of solution A
0.25 mg/l solution
ml
0.50 mg/l solution
ml
0.80 mg/l solution
ml
1.00 mg/l solution
ml
Measure the absorbances of the calibration solutions at 285.2 nm and 0.7 nm bandwidth
and determine the calibration curve.
Calculate the Mg content of the powder.
54
DETERMINATION OF ACTIVE INGREDIENTS OF PANADOL EXTRA BY HPLC
Compounds:
Paracetamol
500 mg
Caffeine
65 mg
in each tablet
Background
HPLC (high-performance liquid chromatography), one of the most frequently used
analytical techniques in the pharmaceutical industry, is suitable for the separation,
identification and quantitation of the soluble components of a solution. The sample solution
(an appropriate solvent is used) is injected onto a adsorbent layer (chromatographic column).
The components of the sample adsorb on the surface of the column. The properties of the
solvent and the compound influence the elution through the column. The eluted compounds
are most often detected on the basis of their light absorption. The retention time is defined as
the time when the compound appears at its maximal concentration in the chromatogram. The
retention time depends on various factors, e.g. the analyzed substance, the chromatographic
column, the solvent and the pressure.
The HPLC separation of substances depends on the phenomena of absorption of the
analyzed compounds and the chromatographic columns. The traditional normal-phase polar
side-chains of silica gel columns adsorb polar molecules, particularly when an apolar solvent
(moving phase) is used. Polar-polar interactions can therefore be strengthened by the
application of an apolar solvent. Elution from the column (stationary phase) occurs as a
function of the polarities of the molecules. Less polar compounds are eluted from the silica gel
column earlier, as their interaction with the column is weaker.
The elution can also be influenced by the composition of the solvent. In gradient
elution, two solvents with different polarities are mixed and used as the moving phase. If the
ratio of the solvents is changed, the adsorption interactions between the molecules and the
column are also changed. At the beginning of the separation, a reverse polarity solvent is used,
which promotes adsorption. The solubilities of polar compounds are lower in apolar solvents,
and polar compounds preferably bind to the polar silica gel column. In contrast with the polar
molecules, apolar molecules do not adsorb on the column, and their elution is faster. If the
proportion of the more polar component of the solvent is increased, the polar molecules will
also be eluted from the stationary phase.
Most drug molecules are apolar, and rather hydrophobic, and reverse-phase
chromatography is therefore used for their separation. In reverse-phase chromatography,
alkyl/aryl groups are covalently attached to the traditional silica gel to form an apolar surface.
Apolar compounds will bind to this with higher affinity, and polar ones with less or no affinity,
and the polar compounds will therefore elute faster from the column.
The current Pharmacopoeia accepts several reverse-phase columns. The names of the
columns reflect the alkyl/aryl groups, e.g. octyl (C8), octadecyl (C18) or phenyl (C6H5). The
type of the column, its length and diameter, and the particle size must be indicated when a
chromatographic separation is performed. The composition of the solvent, the gradient, the
applied pressure, the flow rate and the temperature are all important parameters of this
separation technique.
55
Preparation of calibration solutions
Two calibration stock solutions should be prepared. For Standard1Stock, weigh 100.0
mg of paracetamol and 13.0 mg of caffeine with analytical accuracy into a 50.0-ml volumetric
flask. For Standard2Stock, weigh 105.0 mg of paracetamol and 15.0 mg of caffeine with
analytical accuracy into another 50.0-ml volumetric flask. Dissolve the compounds in
approximately 30 ml of solvent (90v/v% 50 mM phosphate buffer pH 6.3; 10 v/v% acetonitrile)
then make the solutions up to volume with the same solvent. Record the accurate weights and
concentrations.
Dilutions: 1.5-ml aliquots of stock solutions are pipetted into separate 10.0-ml
volumetric flasks and then made up to volume with the earlier solvent. After homogenization,
pour the diluted Standard1 and Standard2 solutions into separate sample flasks. Calculate the
concentrations of the stock solutions and the diluted standard solutions in mg/ml with fourdecimal accuracy, and record the results in the table below:
Standard1
Paracetamol
Weight (mg)
Concentration of stock
solution (mg/ml)
Concentration of diluted standard
solution (mg/ml)
Caffeine
Standard2
Paracetamol
Caffeine
Preparation of the sample
Composite samples are analyzed, not individual samples, see below. So the goal is the
determination of the content of the active ingredient, not uniformity analysis. Put therefore, e.g.
7 tablets, in the case of six students are attending the course, directly from the blister into a
mortar. Grind the tablets in the mortar and homogenize the powder. Grinding is necessary
because film tablets are used for the analysis and the grinding makes the active ingredients are
more accessible for the solvents. Tare a 50-ml beaker on an analytical scale. Weigh 0.697 g ±
0.001 g of the powder. This is approximately the weight of one tablet. Record the accurate
weight of the sample. Dissolve the active ingredients (paracetamol and caffeine) in a beaker
with the earlier solvent (90 v/v% 50 mM phosphate buffer pH 6.3; 10 v/v% acetonitrile) by
shaking the sample. After the shaking, pour the mixture into a 50.0-ml volumetric flask through
a funnel, and then rinse the beaker with a small amount of solvent and finally make up the
volume. The sample will be turbid, containing a flocculent precipitate, as not all of the
components of the tablet are soluble in the solvent. After the larger particles have sedimented,
pipette 300 µl of stock solution from the uppermost part of the solution into a 10.0-ml
volumetric flask and make it up to volume with the solvent. Homogenize the sample solution.
56
The sample solution should be filtered before the HPLC measurement in order to remove the
larger particles and contamination and to avoid clogging the instrument. Adjust the filter onto
the 10-ml syringe (Millipore PVDF 0.45 µm). Pour the sample into the syringe and push it
through the filter. Discard the first 7 ml. The remaining 3 ml is used for the measurement, and
is filtered into the sample flask. The sample is ready for the analysis.
57
COMPLEXOMETRIC TITRATIONS
During complexometric titrations, those standard solutions are used of compounds that
form stable complexes with metal ions. A specific feature of the reaction is the formation of
coordination complexes containing covalent bonds. The central metal ion is complexed by the
ligands of the standard solution. Many metal ions can form six coordinate bonds, and therefore
six electron-rich donor atoms are required for complex formation. The disodium salt of
ethylenediaminetetraacetic acid (EDTA) is one the most commonly used standard solutions in
complexometric titrations. EDTA has two acidic protons.
Ethylenediaminetetraacetic acid disodium salt
EDTA
The complexes of EDTA contain a metal ions. The complex formation must be fast and
quantitative if the reaction is to be used for volumetric analysis.
The EDTA complexes are usually formed rapidly, and they are stable if the central
metal ion is not a monovalent cation (e.g. an alkali metal ion), but their stability does depend
on the pH of the solution. Buffer solutions are therefore always used during complexometric
titrations in order to preserve the stability of the complex at the optimal pH. The stability of
the complex also depends on the charge of the metal. The higher the elementary charge, the
stronger the complex. Metal ions with an elementary charge 3 or 4 can be titrated in strongly
58
acidic medium (pH 2-3). During the titration of divalent transition metal cations, the pH should
be kept between 5 and 6. Hexamethylenetetramine (methenamine or urotropine) is used to
adjust the pH in this case. The complexes of divalent alkaline earth cations are the most
sensitive to protonation, and they are therefore titrated at pH~10. NH4OH/NH4Cl buffer is
suitable for this purpose. The pH~10 buffer pH≈10 is made in the following way: 50 g of
NH4Cl is dissolved in 400 ml of 25% NH4OH solution, which is then made up to 1000 ml. 5
ml of this solution is added to 100 ml of sample. Approximately 2 g of solid urotropine is
necessary to ensure pH 6.
A standard disodium edetate solution can be prepared by direct weighing only if the
material has first been dried at 80 °C for 1 h. Standardization depends on the application. Ca2+
or Zn2+ salts are usually used to determine the factor. H4EDTA is not hygroscopic, but its
solubility in water is limited.
The complexometric indicators are dyes that undergo color changes in the presence of
certain metal ions. The end-point of the titration is reached when the metal has been completely
displaced from the indicator; a constant color is therefore seen at the equivalence point. The
complexometric indicators participate in protonation equilibrium processes and their colors
depend on the pH of the solution. Eriochrome black T is used as indicator between pH 7 and
the pH where the violet color of the metal ion complex turns blue at the equivalence point due
to the presence of the single protonated blue form. (A double protonated red form exists at
pH<6, but it is not not used as a complexometric indicator because its metal complexes are
also red. Eriochrome black T is orange in its nonprotonated form at pH>12.)
Some complexometric indicators are not stable in solution, and are therefore triturated
with indifferent salts such as NaCl or KNO3 are usually used to make a solid mixture. The
mixture usually contains 1% of the indicator and 0.1-0.2 g of it is used during the titration of a
sample.
ION
INDICATOR
pH
END-POINT OF THE
TITRATION
FORM
DISTURBING
IONS
Mg2+ Eriochrome black T
10
violetblue
1% (KNO3)
Ca2+ Murexide
>12
redviolet
1% (KNO3)
Bi3+ Thymol blue
1-3
blueyellow
1% (KNO3)
Fe3+
Hg2+ Thymol blue
6
blueyellow
1% (KNO3)
Fe3+
Al3+ Dithizone
4
greenish-bluereddishviolet
0.025%
(solution)
1-3
pinkyellow
solution
Ca2+ Calconcarboxylic acid >12
violetblue
solution
Bi3+ Xylenol orange
59
Al3+, Fe3+
O NH4
O
R
N
R
N
N
O
O O
N
R
N
Murexide
R
S
N
N
N
H
Dithizone
60
NH
O
61
62
PULVIS NEUTRACIDUS
(PULV. NEUTRACID.)
ANTACIDUM. ADSTRINGENS.
Composition Calami rhizoma pulvis
:
1.0 g
Frangulae cortex pulvis
1.0 g
Natrii hydrogenocarbonas
8.0 g
Bismuthi subnitras
14.0 g
Magnesii subcarbonas
16.0 g
Total mass:
40.0 g
40 doses of divided powder
Preparation of the sample
The sample needs special treatment before the analysis. The inorganic compounds in
the sample are made ready for the analysis during this preparation. Certain components of the
powder mixture, e.g. anthraquinone compounds in the frangulae cortex (glucofrangulins,
frangulins or frangula emodin) form strong complexes with the metal ions in the sample, and
would therefore disturb the analysis of the inorganic ions (Mg2+ and Bi3+). The sample must
be heated to destroy its organic components.
63
Heating:
0.2500 g of sample is weighed with analytical accuracy into a porcelain jar. The sample
is heated carefully and completely annealed. 10.0 ml of 30% HNO3 is added in small portions
to the cooled sample until it has dissolved, and the solution is then washed it into a 100.0-ml
volumetric flask with water.
The Bi3+ and Mg2+ contents of the sample are analyzed by complexometry with sodium
edetate standard solution in the presence of indicators. The analysis of these ions is possible in
the same sample because the bismuth edetate complex is stable in acidic medium, whereas
while the magnesium complex is stable in slightly basic solution.
Determination of bismuth content:
20.00 ml of the stock solution is diluted to 100 ml in a 200-ml flask. Add 5-6 drops of
xylenol orange indicator to the sample and titrate it with 0.01 M sodium edetate solution until
the solution turns from pink to yellow.
1 ml of 0.01 M sodium edetate is equivalent to 2.09 mg of Bi.
Determination of the magnesium content:
Dilute 20.00 ml of stock solution with 70 ml of water in a 200-ml flask and then add 6
M NH4OH solution (about 2 ml) to neutralize the sample; it turns cloudy. Dissolve 0.25 g of
NH4Cl in the sample solution; then mix it with 10 ml of 6 M NH4OH solution (pH~10). Add
0.1 g of Eriochrome black T and titrate it with 0.02 M sodium edetate standard solution until it
turns from magenta to blue. The solution should keep its blue color for 5 min.
1 ml of standard 0.02 M sodium edetate is equivalent to 0.8064 mg of MgO.
Notes:
Examine the porcelain jar carefully before the experiment and look for fine cracks
(cracked jars should not be used). Set the burner to jet lance, as otherwise the heating process
is not successful. Distribute the powder evenly at the bottom of the pot. By the end of the
heating, the sample is dark-red, but this changes to yellow when the sample cools down. The
hot ceramic pot should not be put directly onto the bench, but also onto an asbestos surface.
Mix the sample with a glass rod when it is being dissolved in HNO3 because this is a slow
process.
During the analysis of Bi, the indicator turns fro pink to yellow at the equivalence point
when only a small drop is added.
During the analysis of Mg the change in color of the indicator from magenta to blue is
difficult to see. Use an overtitrated sample for comparison. The blue color should be stable for
5 min.
64
SUSPENSIO ZINCI AQUOSA
(SUSP. ZINC. AQUOS.)
DERMATOLOGICUM.
Composition:
Zinci oxydum
20.0 g
Talcum
20.0 g
Glycerinum
10.0 g
Ethanolum 70%
10.0 g
Aetheroleum menthae piperitae
X gtt
Solutio acidi borici 2% FoNo VII.
40.0 g
Total mass:
100.0 g
Determination of the zinc content of a sample
The Zn content of a sample is determined by complexometric analysis. The suspension
should be shaken very well before the measurement in order to analyze a homogeneous sample.
Approximately 0.5 ml (0.500-0.600g) of suspension is used for the titration. The sample is
transferred to a 50-ml beaker with a Pasteur pipette and its accurate weight is checked. 10 ml
of R diluted acetic acid is added to the sample. The mixture should be shaken until it turns
opalescent (about 1 min).
Zn is dissolved in a weak acid, and free Zn2+ ions are obtained. Other components in
the sample are not soluble in acetic acid, and therefore do not disturb the titration.
The solution is washed into a 250-ml titration flask with 100 ml of R-water. 50 mg of
xylenol orange trituration and 2 g of hexamethylenetetramine are then added to the sample.
Immediately after the hexamethylenetetramine has completely dissolved, the pinkish-violet
solution is titrated with 0.1 M sodium edetate standard solution until the solution turns yellow.
Calculate the percentage ZnO content of the sample. Record the result with two-decimal
precision.
1 ml of 0.1 M sodium edetate standard solution is equvivalent to 8.14 mg of ZnO.
65
ARGENTOMETRIC ANALYSIS
Argentometric titration is a special type of volumetric analysis. The standard solution
in argentometry is AgNO3, which can be stored for a long time when it is protected from light.
The simplest method in argentometry is precipitation titration, when potassium thiocyanate
(KSCN) (potassium rhodanide) or NH4SCN is used as a secondary standard solution. (Ph. Eur.
recommends only standard NH4SCN solution.) Standard thiocyanate solutions slowly
decompose, so they should be checked before use. As an example, during the determination of
I- an excess of AgNO3 is added first to the sample, and then AgNO3 is titrated with KSCN. The
SCN- ion forms a precipitate of AgSCN. If Fe3+ is added to the sample as an indicator, the red
color of Fe(SCN)3 appears at the equivalence point of the titration (Volhard method).
The proton of acidic N-H bonds can be replaced by Ag+ and non-ionic silver
compounds are formed. Two types of measurements are possible in this case. When
sulfadimidine is analyzed, direct titration is performed with standard AgNO3 solution and
CrO4- as indicator. The silver compound of sulfadimidine is not ionic at the equivalence point,
and the excess Ag+ forms a brown AgCrO4 precipitate with CrO4-.
During the analysis of theobromine (Boie method), standard AgNO3 solution is added
to the sample and the non-dissociating silver compound of theobromine and an equivalent
amount of HNO3 are formed. The HNO3 is titrated with standard alkaline solution. This method
is a combination of argentometry and acidimetry.
The acidbase titrations can be followed potentiometrically, when the pH is checked.
After the titration curve has been plotted, the equivalence point can be determined graphically.
The first derivative curve should be used when the equivalence point is not sharp enough.
Conductometry is a convenient method with which to follow precipitation titrations.
66
SPARSORIUM SULFABORICUM
(SPARS. SULFABOR.)
DERMATOLOGICUM.
Composition: Sulfadimidinum
5.0 g
Acidum boricum
5.0 g
Total mass:
10.0 g
CH3
O
O
N
S
N
H
N
CH3
H2N
Sulfadimidine
Determination of the sulfadimidine content of a sample:
Accurately weigh 0.3500 g of powder and dissolve it in acetone with gentle heating (it
takes approximately 15 min). The dissolution of sulfadimidine in acetone is slow, and the
sample must therefore be gently heated. It should not be boiled as acetone is flammable. 20 min
is necessary for complete dissolution, and the solution is then cooled to room temperature.
Next add 0.5 g of MgO and 2 drops of 10% K2CrO4 solution, when a yellow suspension
is obtained. It is practical to use only a small amount of indicator (1-2 drops) because the color
change can be observed more easily when light colors are used.
Five min later, the reaction mixture is titrated with standard 0.1 M AgNO3 solution. The
mixture is shaken thoroughly after each drop of standard solution. The titration should be
carried out slowly, especially close to the end-point. It is recommended to wait 10-15 sec after
the addition of each drop of standard solution. The end-point of the titration has not been
reached if the solution turns back to yellow and the color of the precipitate is still white. Close
to the end-point of the titration, it is recommended to wait a little after shaking. The yellow
mixture turns salmon-pink at the end-point of the titration.
1 ml of standard 0.1 M AgNO3 is equivalent to 27.833 mg of sulfadimidine. Record the
result in g with four-decimal precision for an average weight of 10 g.
67
REDOX TITRATIONS
The application of redox reactions in volumetric analysis is called redox titration. We
will discuss oxidimetry, when the standard solution is an oxidizing agent, while in case of
reductometry the standard solution is a reducing agent.
The most important oxidimetric methods:

permanganometry
(the standard solution is KMnO4)

chromatometry
(the standard solution is K2Cr2O7)

cerimetry
(the standard solution is Ce(SO4)2 or (NH4)2Ce(SO4)3)

bromatometry
(the standard solution is KBrO3)
The most important reductometric method is ascorbinometry, where the standard
solution is ascorbic acid.
A special case of redox titration is iodometry, which can be used for either oxidimetry
(iodimetry) or reductometry (iodometry). The standard solutions of iodometric methods are I2
and Na2S2O3 solutions.
Bromatometry is sometimes combined with iodometry. After the addition of KBr and standard
KBrO3 solution, excess Br2 is liberated. Br2 reacts with I- and I2 is formed. The I2 can be titrated
with standard Na2S2O3 solution.
The equivalence point of the redox titration can be detected by indicator dyes such as
ferroin, by the reaction of I2 and starch, or simply through the disappearance of the color of I2.
Redox titrations can be followed by potentiometry or biamperometry.
The standard solution of bromatometry is KBrO3 solution, which can be prepared
precisely by direct weighing. Standardization is not necessary as it is very stable. 0.033 M (0.1
N) and 0.02 M stock solutions are usually made directly; dilutions of stock solutions are also
used. The standard solution of bromatometry can be used as a direct oxidizing agent or for the
preparation of bromine solutions.
Direct bromatometric analysis involves the titration of ascorbic acid with Br2 is formed
when Br- ions are added to the reaction mixture, according to the following reaction:
BrO3- + 5 Br- + 6 H+ = 3 Br2 + 3 H2O
The Br2 formed reacts with ascorbic acid. The reaction is fast; dehydroascorbic acid is formed.
68
Back-titration is used when the reaction with Br2 is not fast enough for the analysis. Brand KBrO3 are added to the sample to form Br2. The reaction is allowed to go completion. The
excess Br2 is titrated iodometrically.
Organic compounds are brominated in addition or substitution reactions. Alkenes react
with Br2 in addition reactions. Br2 is volatile, so special brominating flasks are used.
Br
Br2
Br
This reaction is used for the analysis of unsaturated fatty acids. The iodine/bromine
index gives information about the number of double bonds in the sample. The analysis of
hexobarbital is also based on a Br2 addition reaction.
Bromine substitution is specific for highly reactive aromatic compounds (containing
first class substituents). Phenols, aminobenzenes, salicylic acid and anthranilic acid are
analyzed in this way.
In the analysis of salicylic acid by the Koppeschaar method, tribromophenol bromine
is formed by bromine substitution and decarboxylation.
OH
OH
CO2H
Br
Br
+ 3HBr +CO2
+ 3Br2
Br
2,4,6-tribromophenol
Salicylic acid
O
OH
Br
Br
Br
Br
+Br2 ,-HBr
+2HI, -I2-HBr
Br
Br
Br
Tribromophenol bromine
69
Tribromophenol bromine reacts similarly to elementary Br2 with I- ions; when the Br2
is titrated iodometrically, three bromines are involved in the reaction.
Acetylsalicylic acid cannot be determined by bromination as it does not contain a free
aromatic hydroxy group and it reacts slowly with Br2. When a longer reaction time is applied,
the acetyl group is hydrolyzed and not acetylsalicylic acid, but salicylic acid is determined.
Bromatometry is therefore not used for the analysis of acetylsalicylic acid.
Several standard solutions are used in cerimetry. Ce(SO4)2, (NH4)2Ce(SO4)3 and
Ce(NH4)2(NO3)6 standard solutions are all highly acidic.
The hydrolysis of Ce salts is blocked when the pH is low. H2SO4 is usually used to
decrease the pH, such cerimetric standard solutions being highly stable. Standard Ce(SO4)2
solution is prepared by dissolving Ce(SO4)2 in dilute H2SO4. Iodometry is used to standardize
Ce(SO4)2 volumetric solutions. I2 is titrated when Na2S2O3 solution is used after the addition
of KI.
Standard (NH4)2Ce(SO4)3 solution is prepared by dissolving the compound in dilute
H2SO4, and iodometry is used to standardize the solution. (NH4)2Ce(SO4)3 exists in two forms:
anhydrous and hydrated; the anhydrous (NH4)2Ce(SO4)3 is used to prepare more accurate
standard solutions.
Ferroin indicator is used in cerimetry where three phenanthroline ligands coordinate an
Fe or Fe3+ ion.
2+
Cerimetric oxidation is most often used for the measurement of Fe2+. The Fe content
of Fe(II) gluconate is determined by the titration of standard (NH4)2Ce(SO4)3 solution in the
presence of ferroin as indicator.
The standard cerimetric solutions are strong oxidizing agents in aqueous medium. The
following chemical equation describes the reaction:
4 Ce4+ + 2 H2O  4 Ce3+ + 2 “O··” + 4 H+
70
Two Ce4+ ions as oxidizing agent are equivalent to one nascent oxigen “O··” (oxygen radical).
This is a free radical reaction, where H2O accepts an electron, while Ce3+, a hydroxyl
radical (HO·) and a proton are formed.
Ce4+ + H2O  Ce3+ + [H2O·]+
[H2O·]+  HO· + H+
2 HO·  H2O + “O··”
The cerimetric determination of aminophenazone by the method of Rózsa is based on the
above-mentioned reaction.
Iodometric methods are divided into two different groups. The first type is when I2containing solutions are titrated with S2O32- (iodometry), and the second type is when reducing
agents are titrated with standard I2 solution (iodimetry). Standard S2O32- solutions are made
directly from powder, which is dissolved in freshly boiled distilled water (CO2 free) together
with a small amount of (0.2 g) Na2CO3. I2 standardized with KBrO3 is used to determine the
titer of the S2O32- solution.
BrO3- + 6 I- + 6 H+  Br- + 3 H2O + 3 I2
S2O32- is one of the most unstable standard solutions. Its titer changes after preparation,
first increasing slowly, and then decreasing gradually. It should therfore be stored for several
days, when it reaches its final titer. If it is necessary to use the solution immediately after
preparation, it must be standardized immediately before the titration. It must be borne in mind
that this titer cannot be used in later measurements. The solution cannot be used for volumetric
analysis if it contains colloidal sulfur precipitate. Such precipitation of sulfur is a result of
71
bacteria present in the solution. To prevent bacteria form growing in the solution, 1-2 ml of
amyl alcohol is added to the volumetric solution before it is made sup to the final volume.
I2 is not soluble in water; it is therefore dissolved together with KI, when KI3 is formed,
a complex which dissolves very well in water (e.g. to prepare a 0.5 M standard solution, 127 g
of I2 and 200 g of KI is dissolved and diluted to a final volume of 1000 ml)
I2 + KI = KI3
(K = 710)
Standard I2 solutions are kept in the dark and their titers are determined with Na2S2O3.
Reducing agents can be analyzed with standard I2 solutions if they undergo oxidation
instantaneously, and excess I2 is not necessary for the reaction. Standard KIO3 solution and
back-titration of the excess I2 is used if the above requirements are not fulfilled. I2 is formed
from KIO3 and I2- according to the following reaction:
IO3- + 5 I- + 6 H+ = 3 I2 + 3 H2O
I2 reacts with S2O32- to form tetrathionate ions (S4O62-):
I2 + 2 S2O32-  2 I- + S4O62I3- + 2 S2O32-  3 I- + S4O62The amylose component of starch (water-soluble starch) is used as an indicator in
iodometry. Amylose consists of 50-100 glucose monomers, which forms a spiral. I113- chains,
consisting of 3 I3- + I2, enter these helices, and blue iodine–starch is formed. The color is so
intense that it can be detected even at even I2 concentrations as low as 1·10-5 M. The solution
is kept acidic when iodine–starch is used as indicator. Starch should be added just before the
equivalence point of the titration because iodine–starch can precipitate and its reaction with
S2O32- becomes very slow. The iodine–starch indicator does not function when the ethanol
concentration is >50%. I- ions are necessary for the formation of iodine–starch. I2 is soluble in
chlorinated hydrocarbons such as chloroform (CHCl3). Another typical indication method is
when several ml of choloroform is added to the sample. Iodine dissolves in chloroform to give
a purple color. This purple color disappears at the equivalence point of the titration. Vigorous
shaking of the sample is necessary during the titration!
Excess Br2 can be determined by iodometric titration with standard S2O32- solution after
bromination reactions according to the following equation:
Br2 + 2 I-  I2 + 2 BrI2 + 2 S2O32-  2 I- + S4O62-
72
As an example, excess Br2 is determined after Br2 addition in the case of hexobarbital,
or bromine substitution in the case of salicylic acid. I2 does not react with olefins or aromatic
compounds, and indirect analysis therefore is used in these cases.
Strong reducing agents can be titrated with standard I2 solution (iodimetry). The sulfur
dioxide (SO2) liberated from noraminophenazone sodium salt by acidic hydrolysis is measured
by standard I2 solution.
SO32- + I2 + H2O = SO42- + 2 I- + 2 H+
Ascorbic acid can be titrated with I2 solution directly.
Other redox techniques, such as permanganometry, chromatometry and
ascorbinometry, are not discussed in detail here, as they are not used in the pharmacopoeia.
Biamperometry (dead-stop titration) is especially useful for following redox titration
reactions. Electric current flows while both the oxidized and reduced forms are present. The
current stops at the equivalence point of the titration, when only either the oxidized or the
reduced form is present (e.g. the reduced form is completely oxidized). When only one form
is present at the beginning of the analysis, current flows only when the other form appears in
the solution. The current therefore increases at the beginning of the titration, and then slowly
decreases as the concentration of the first form decreases. A bell-shaped titration curve is
observed. If the redox reaction of the analyzed sample is reversible electrochemically, e.g. Fe2+
is titrated cerimetrically, the current increases after the equivalence point is reached. The
equivalence point is the minimum in the titration curve. Biamperometry is used for water
analysis by the Fischer method.
73
SUPPOSITORIUM ANTIPYRETICUM PRO INFATE VEL PRO PARVULO
(SUPP. ANTIPYRET.)
ANTIPYRETICUM. ANALGETICUM.
Composition: Aminophenazonum
0.45 or 0.9 g
Adeps solidus compositus
qu.s.
6 suppositories
Background:
During the quantitative analysis, the double bond of the pyrazolone ring is oxidized to
a diacyl hydrazine compound (“dioxypyramidone”) by four Ce4+. The equivalent weight is
therefore one-fourth of the molecular weight.
Water is oxidized by Ce4+ ions as follows
Ce4+ + H2O →
[H2O]+
Ce3+ + [H2O]+
→
2 HO →
HO + H+
H2O + O
The pyrazolone ring is oxidized to dioxypyramidon by nascent oxygen (O).
Aminophenazone is a strong carcinogenic agent because nitrosodimethylamine may be
formed in the living body during its metabolism.
74
H3C
O
N
N
H3C
Nitrosodimethylamine
It is not registered in Ph.Eur.
Determination of the aminophenazone content of a sample:
Rózsa method: A suppository is melted in a beaker and 0.2-0.3 g of melted sample is
weighed with analytical accuracy into a 100-ml titration flask. 10.0 ml of 15% of H2SO4 is
added to the sample and the mixture is heated to about 40 °C to dissolve the aminophenazone.
The sample is allowed to cool to room temperature, 15 ml of distilled water and 1 drop of ferroin
indicator are added to the solution, and titrated with standard 0.1 N (0.1 M) Ce(SO4)2 solution.
The orange color of the sample turns green at the end-point of the titration.
The titration should be continuous without any disruption. The color of the sample may
turn light-green before the end-point, but it may turn back to orange during intensive shaking.
The end-point may then be reached by the addition of 1-2 more drops of standard solution. The
green color should persist for half a minute. Parallel titrations should be performed, because the
sample may not be homogeneous.
1.00 ml of standard 0.05 M Ce(SO4)2 solution is equivalent to 2.891 mg of
aminophenazone. The final result should be calculated in mg with two-decimal precision. The
average weight of the sample is 1.2 g.
75
INJECTIO ALGOPYRINI 50%
(INJ. ALGOPYRIN 50%)
ANALGETICUM.
Background:
The sodium methylsulfonate group in metamizole sodium salt (noraminophenazone) is
hydrolyzed in acidic medium to give 1-phenyl-2,3-dimethyl-4-methylpyrazolone,
formaldehyde and SO2.
SO2 + H2O
H2SO3
H2SO3
HSO3- + H+
HSO3-
SO32- + H+
SO32- + I2 + H2O
SO42- + 2 I- + H+
Quantitative analysis of metamizol sodium salt:
0.4000 g (about 11-12 drops) of material is weighed into a 50-ml Erlenmeyer flask with
a glass stopper. Cool the flask, add a cold mixture of 20 ml of methanol and 3 ml of 50% H2SO4
and then titrate the mixture with standard 0.05 M I2 solution. N.B Keep the temperature below
15 °C (ice-cold water-bath). Do not shake the mixture vigorously as SO2 is volatile.
According to the Ph.Eur., under continuous temperature control (ice-cold water-bath)
after the addition of 0.01 M HCl metamizole sodium is titrated with 0.05 M I2 solution, with
starch as indicator, until the solution turns blue and the color persists for 1 min.
1 ml of standard 0.05 M I2 solution is equivalent to 16.67 mg of water-free
noraminophenazone sodium mezylate/metamizole sodium. The density of the injection at 20 ºC
is 1.1778 g/ml. Calculate the percentage of metamizole in the sample in g / 100 ml with twodecimal precision.
Notes:
The quantitative assay is carried out by the addition of 1-2 drops of alcoholic methylene
blue and the clear blue solution is then titrated with I2 until it turns emerald-green.
76
ACIDBASE TITRATIONS
Acidbase titrations are important in pharmaceutical analysis. Pharmaceutical
compounds frequently contain weak acidic or weak basic groups and their analyses are usually
unique.
The titrations are not always perfomed in aqueous solutions, but often in aqueous
alcoholic or alcoholic medium. Very weak bases are analyzed with a mixture of glacial acetic
acid and HClO4 in nonaqueueos medium.
An acid–base titration curve is a graph of pH against the volume of titrant added during
the titration. The shape of the curve is specific for the analysis. The end-point of the titration
is the neutralization point, the inflexion point (the steepest slope) in the curve.
Dyes are usually used to indicate the equivalence point of acid–base titrations. An acid–
base indicator is a compound whose protonated and deprotonated forms have different colors.
The pH change is indicated by the color change of the indicator. A small error is introduced
because the indicator also consumes titrant, and it should therefore be used in only a small
quantity. Moreover the color change can be detected more easily when a small quantity of
indicator is used. The choice of the indicator depends on the equivalence point of the titration.
The pH range in which the color of the chosen indicator changes should be really close to that
at the end-point of the titration. The indicator exists in both dissociated and nondissociated
forms at the equivalence point of the titration, and therefore a transition color is visible. Visual
detection of the transition color is difficult, because it depends on a number of factors. A color
change is noticed by the human eye if one form becomes dominant, which means at least a 10fold excess. A transition interval is therefore indicated, not a transition point. Mixed indicators
are sometimes used to make the color change easier to detect. In most of these cases, the
original color turns to its complementary color. In one ideal case, red turns to green, when the
transition color is gray. The complementary color change can be achieved through the addition
of dyes such as methylene blue instead of a second indicator.
The acid–base feature of the indicators also depends on the media and the temperature.
To decrease the possibility of errors during the analyses, the recommended indicator should be
used at very close to room temperature.
The features of the indicators are influenced by various factors. The transition of an
acidic indicator is shifted in the direction of acidic pH when the temperature increases, while
the transition of a basic indicator is shifted in the direction of basic pH. Organic solvents such
as alcohols shift the transition of acidic indicators to a more basic pH and of basic indicators
to a more acidic pH. Colloid solutions adsorb the indicator and change the color transition. The
behavior of indicators is influenced by the presence of CO2 and NH3 possibly dissolved from
the atmosphere. Boiling the sample before the equivalence point may be a suitable way to
remove CO2 in the case of basic indicators such as phenolphthalein or thymolphthalein, or to
remove NH3 in the case of acidic indicators. The sample should then be cooled back to room
temperature before the titration is completed.
Standard glacial acetic acid–HClO4 solution is used for the analysis of very weak bases,
and the titration is performed in glacial acidic acid medium. Such weak bases include lidocaine,
benzocaine, procaine, xanthine derivatives such as theophylline, theobromine, etc., and certain
sulfonamides that contain primary or secondary amino groups.
77
INDICATOR
TRANSITION
INTERVAL
pKI
COLOR
ACID
BASE
Methyl violet
0.1-3.2
yellow
violet
Cresol red
0.2-1.8
red
yellow
Thymol blue
1.2-2.8
red
yellow
Dimethyl yellow
2.4-4.0
3.45
red
yellow
Bromophenol
blue
3.0-4.6
4.06
yellow
violet
Methyl orange
3.2-4.4
3.76
red
orange
Bromcresol green
3.9-5.4
4.90
yellow
blue
Methyl red
4.2-6.2
4.96
red
yellow
Bromothymol
blue
6.0-7.6
7.3
yellow
blue
Neutral red
6.8-8.0
7.4
red
yellow
Phenol red
6.8-8.2
7.72
yellow
red
Cresol red
7.2-8.8
8.08
yellow
red
Thymol blue
8.0-9.2
8.82
yellow
blue
Phenolphthalein
8.0-10.0
9.5
colorless
red
Thymolphthalein
9.4-10.6
9.7
colorless
blue
Tropaeolin 00
11.0-13.0
yellow
orange
MIXED INDICATORS
PROPORTION
TRANSITION
PH
COLOR
ACID
BASE
Methyl redBromocresol green
1:3
5.1
orange
green
Methyl red –
Methylene blue
1:1
5.3
violet
green
Phenolphthalein Methylene green
1:2
8.8
green
violet
Standard glacial acetic acid–HClO4 solution is prepared from a mixture of the most
concentrated (70%) HClO4 solution and glacial acetic acid. Acetic anhydride is added to the
mixture in an amount equivalent to the water in the solution. As an example standard 0.1 M
glacial acetic acid– HClO4 solution is prepared from 8.5 ml of HClO4, 900 ml of glacial acetic
acid and 30 ml of acetic anhydride, and the mixture is diluted to 1000 ml with glacial acetic
acid. The water content of the standard solution is checked 24 h later (e.g. the Fischer method)
and set to between 0.1% and 0.2% with water or acetic anhydride and the mixture is left to
stand for another 24 h. The standard solution must not contain acetic anhydride, which would
78
react with primary or secondary amines. The solution is standardized with potassium
hydrogenphthalate in the presence of crystal violet as indicator.
The temperature of the solution is important during the standardization because of
differences in thermal expansion. When an analysys is carried out at a temperature that is
different from the temperature of standardization, the volume should be corrected by using the
following formula: VCorr=V[1+(T1-T2)·0.0011], where V is the volume measured in analytical
titration at T2, while T1 is the standardization temperature. Crystal violet (yellow to violet),
cresol green (yellow to red) and methyl violet can be used as indicators with standard glacial
acetic acid–HClO4 solution.
Tetrabutylammonium hydroxide is used as nonaqueous basic standard solution for the
titration of acids. Its standard 1 M solution is commercially available.
Its toluene solution is prepared from tetrabutylammonium iodide with Ag2O in the
presence of methanol, and then diluted with toluene. Benzoic acid is used in the presence of
thymol blue as indicator for standardization directly before use.
This is a very sensitive standard solution. It should be kept away from light and
moisture. It can be used for potentiometric analysis acids of different strengths, inorganic acids,
carboxylic acids and phenols in the same sample.
79
SPIRITUS IODOSALICYLATUS
(SPIR. IODOSALIC.)
ANTIMYCOTICUM. ANTISEPTICUM
Composition: Potassii iodidum
0.10 g
Acidum salicylicum
0.90 g
Solutio iodi alcoholica
6.00 g
Ethanolum 70%
23.00 g
Total mass:
30.00 g
Ingredients of Solutio iodi alcoholica (Ph. Hg. VII.)
Composition: Potassium iodatum
4.0
g
5.0
g
Aqua destillata
10.0
g
Spiritus contentratissimus
81.0
g
100.0
g
Iodum
Total mass:
Determination of iodine content:
Weigh 4.0000 g (4.5 ml) of substance into an Erlenmeyer flask with a glass stopper.
Add 10 ml of cold freshly distilled water and titrate the solution with 0.1 M Na2S2O3 solution
in the presence of 2 drops of starch solution.
Spiritus iodosalicylatus is volatile at room temperature. First tare the Erlenmeyer flask
(together with the glass stopper), remove it from the scale, add the 4.5 ml of substance to the
flask, and replace the glass stopper immediately. Measure it as quickly as possible. Avoid
putting any substance on the side-walls of the flask.
The analysis can be performed without the addition of starch, simply by monitoring the
disappearance of the color of I2. It is recommended to add starch only at the very end of the
titration, when the color of the solution is pale-yellow, and then continue the titration until the
blue color disappears.
1 ml of standard 0.1 M Na2S2O3 is equivalent to 12.69 mg of I2. Calculate the mass of
I2 in 30 g of substance and give the result in mg with two˗decimal precision.
Determination of salicylic acid:
Add 3 drops of phenolphthalein indicator to the solution used for I2 analysis and titrate
with it 0.1 M NaOH solution. The addition of freshly distilled water is necessary so that this
acidbase titration can be carried out properly. During the titration, precipitate of salicylic acid
may be observed, which dissolves on the addition of NaOH. Care must be taken to ensure that
the, salicylic acid precipitate (needle crystals) should be dissolved completely and not left on
80
the walls of the flask. At the equivalence point, the pink color of the solution should persist for
at least 1 min.
1 ml of standard 0.1 M NaOH solution is equivalent to 13.81 mg of salicylic acid.
Calculate the mass of salicylic acid in 30 g of substance and give the result in mg with twodecimal precision.
Determination of potassium iodide content:
This analysis is not included in this semester requirements. Add 5 ml of 1 M H2SO4
and 50 ml of water to the solution used for the analysis of salicylic acid. The mixture is titrated
with 0.1 M AgNO3 solution in the presence of metanil yellow.
NaO3S
N
H
N
N
Metanil yellow
(red at pH 1.5, yellow at pH 2.7)
The AgNO3 is added dropwise at close to the equivalence point until the grayish-blue
color of the solution turns cyclamen red and the dye adsorbed on the surface of the precipitate
is blue. Subtract the volume measured in the I2 analysis from the volume measured in the KI
analysis.
1.00 ml of standard 0.1 M AgNO3 is equivalent to 16.600 mg of KI.
This titration is performed with the addition of phenol red (yellowred transition)
according to Ph.Eur.
81
TEST YOURSELF – SAMPLE TEST QUESTIONS
1. Which of these phenomena is the most important in UV spectroscopy?
a.
b.
c.
d.
e.
Excitation of the rotation of the molecules.
Excitation of the rotation of the substituents.
Excitation of the electron system of the molecules.
Excitation of the outer electrons of light atoms.
Excitation of the inner electrons of atoms.
2. Absorbance is defined by the intensities of the incoming light (I0) and the transmitted
light. Which of the following equations is correct?
a.
b.
c.
d.
e.
A= I0/I
A=log I0/I
A= (I0-I)/I
A= (I-I0)/I
A=(log I0)/I
3. Which wavelength region is official for IR analysis in the Pharmacopoeia?
a.
b.
c.
d.
e.
2.5-15 cm-1
60-208 cm-1
206-560 cm-1
670-4000 cm-1
3800-6000 cm-1
4. Which light source is used in atomic absorption spectroscopy?
a.
b.
c.
d.
e.
A tungsten lamp
A deuterium lamp
A helium-neon laser
A Nernst lamp
A hollow cathode lamp
5. Which data are necessary to calculate the content of an active substance in
spectrophotometric analysis?
a.
b.
c.
d.
e.
The absorbance, the relative absorption coefficient and the length of the cell.
The absorbance and the length of the cell.
The absorbance and the wavelength of the applied electromagnetic wave.
The absorbance and the molecular weight of the active ingredient.
The absorbance, the molar absorption coefficient and the length of the cell.
6. What is contained in the calomel electrode?
a.
b.
c.
d.
e.
Hg/HgCl2
An appropriate pH buffer
Hg/Hg2Cl2/KCl
NaCl solution
Ag/AgCl
82
7. Which of the following are found as two units in a double-beam spectrophotometer?
a.
b.
c.
d.
light source, cell, detector
cell
cell, monochromator, detector
light source, monochromator, detector
8. What happens to the absorption of a compound at the isobestic point?
1.
2.
3.
4.
The absorption ceases.
The absorption increases.
The absorption is unaltered.
The absorption decreases.
a.
b.
c.
d.
e.
Only the 1 is correct.
Only the 2 is correct.
Only the 3 is correct.
Only the 4 is correct.
None of them are correct.
9. Which data are necessary to calculate the relative absorption coefficient of a substance?
1.
2.
3.
4.
5.
The slit width (cm)
The path length (cm)
a.
b.
c.
d.
e.
The concentration of the solution (g/100 ml)
The molecular weight of the substance.
The absorbance of the sample.
Only points 1 and 2 are correct.
Only points 1 and 4 are correct.
Only points 1 and 3 are correct.
Only points 1, 3 and 4 are correct.
Only points 2, 3 and 4 are correct.
10. Which of these statements are correct?
1.
2.
3.
4.
a.
b.
c.
d.
The API of Algopyrin injection is determined at room temperature.
Standard I2 solution is used, which reduces the API.
The sample is shaken slowly to avoid the volatilization of SO2.
The color of the starch indicator disappeares at the end-point of the titration.
Only point 3 is correct.
Only points 1 and 4 are correct.
Only points 1, and 4 are correct.
Only points 2 and 3 are correct.
11. Which statements are incorrect?
1. Spiritus jodosalicylatus is measured quickly into a titration flask to avoid the
evaporation of the alcohol.
2. The salicylic acid content of a sample is determined by acidbase titration with
standard HCl solution.
3. Only a few drops of indicator are used because the acidbase indicator does not utilize
any standard solution.
4. The end-point of the titration is indicated by the appearance of the pink color of
phenolphthalein.
a.
b.
c.
d.
e.
Only points 1 and 2 are incorrect.
Only points 1 and 4 are incorrect.
Only points 2 and 3 are incorrect.
Only points 1, 3 and 4 are incorrect.
Only points 2, 3 and 4 are incorrect.
83
12. Which statements are incorrect?
1. The aminophenazone content of Suppositorium antipyreticum is determined by
cerimetry.
2. Standard Ce2(SO4)3 solution iss used.
3. The aminophenazone ring was opened by nascent oxygen.
4. Indicator is ferroin.
a.
b.
c.
d.
e.
Only point 2 is incorrect.
Only points 1 and 3 are incorrect.
Only points 1, 2 and 3 are incorrect.
Only points 1 and 4 are incorrect.
None of them are correct.
13. 20% of a monochromatic light beam is passed through a solution. What percentage of
the incident light I0 is transmitted?
a.
b.
c.
d.
e.
5%
10%
20%
40%
The given data are not sufficient to determine this.
14. Which one of the following are true? The light absorption of a two-component system is
described by (the absorbance of the components do not affect each other):
a.
b.
c.
d.
AAB=AA+AB
AAB=AA-AB
AAB=log(AA/AB)
TAB=TA+TB
15. Which gas can be used as cooling gas in the case of ICP?
a.
b.
c.
d.
N2
Ar
O2
Ne
16. The Na+ content of an infusion can be determined
1.
2.
3.
4.
5.
a.
b.
c.
d.
e.
on the basis of the absorption of Na+ in VIS (yellow light).
by flame photometry.
by atomic absorption spectroscopy.
on the basis of the yellowish-green absorption of Cl- ions.
by complexometric titration with EDTA.
Only point 1 one is correct.
Only points 2 and 3 are correct.
Only points 1, 2 and 3 are correct.
Only points 2, 3 and 4 are correct.
All of them are correct.
84
17. Which statements are true for the complexometric determination of the cation content
of Pulvis neutracidus?
1. Side-by-side determination of Bi3+ and Mg2+ is not possible.
2. The sample should be ignited before the analysis because of its glucofrangulin content,
which can form complexes with inorganic cations.
3. Bi3+ can be titrated directly after acidic digestion.
4. Eriochrome black T is used in 1% trituration because the indicator utilizes the standard
solution.
a.
b.
c.
d.
e.
Only point 2 is true.
Only points 1 and 3 are true.
Only points 2 and 3 are true.
Only points 2, 3 and 4 are true.
All of the answers are true.
18. Which statements are true as concerns separation techniques?
1. Al2O3, SiO2 or paper can be used as solid-phase absorbent.
2. A reverse-phase column can be obtained by the transformation of SiO2 to silica
chloride.
3. Polar substances pass fastest through a reverse-phase column.
4. A chemical reaction is observed between the analyte and the eluent.
5. Liquids can be used as stationary phase in gas chromatography.
a.
b.
c.
d.
e.
Only points 1, 2 and 3 are true.
Only points 2, 3 and 4 are true.
Only points 1 and 4 are true.
Only points 1 and 5 are true.
Only points 2, 3 and 5 are true.
19. The following units correspond to ppm:
1.
2.
3.
4.
5.
μg/ml
mg/ml
μg/dm3
mg/l
pg/ml
a.
b.
c.
d.
e.
Only points 2, 3 and 5 are true.
Only points 5 is true.
Only points 3 and 5 are true.
Only points 3, 4 and 5 are true.
Only points 1 and 5 are true.
a.
b.
c.
d.
e.
Only points 3 and 4 are true.
Only point 2 is true.
Only point 3 is true.
Only points 2, 3 and 4 are true.
Only points 1, 2 and 5 are true.
20. The following units correspond to ppb:
1.
2.
3.
4.
5.
μg/l
pg/l
ng/dm3
pg/cm3
μg/cm3
85
21. Which statements are true for atomic absorption spectroscopy?
1.
2.
3.
4.
5.
All the elements can be analyzed by this technique in ppm concentration.
Some non-metallic elements can be analyzed by this technique.
It may help the diagnosis of Wilson’s syndrome.
The sample is solvated during the atomization.
0.001% relative precision can be achieved.
a.
b.
c.
d.
e.
Only point 1 is true.
Only points 2 and 4 are true.
Only points 2 and 3 are true.
Only points 1 and 4 are true.
All of the answers are true.
22. Oxidation is experienced when an acetylene-air flame is used for atomic absorption.
How can the problem be solved?
1.
The proportion of acetylene should be
decreased.
2.
3.
The proportion of air should be decreased.
4.
5.
The proportion of fuel gas should be
increased.
The proportion of combustive gas
should be increased.
The proportion of combustive gas
should be decreased.
a.
b.
c.
d.
e.
Only points 1, 3 and 5 are true.
Only points 2, 3 and 4 are true.
Only points 1, 3 and 5 are true.
Only points 2, 3 and 5 are true.
Only points 1, 3 and 4 are true.
23. Which statements are true for the determination of protein concentration?
1. Both UV and VIS spectroscopy can be
used.
2. The applied dyes shift the spectrum to
lower wavelengths.
3. The oxidation of copper ions is utilized
in several techniques.
4. Fluorimetry can be applied.
a.
b.
c.
d.
e.
Only points 1, 2 and 4 are true.
Only points 1, 2 and 3 are true.
Only points 2 and 3 are true.
Only points 2 and 4 are true.
Only points 2, 3 and 4 are true.
24. Which statements are true for the classical titration of Suppositorium antipyreticum?
5. Standard Ce(SO4)2 solution is used that
is diluted in H2SO4 to maintain its
stability.
a. Only points 1, 2 and 4 are true.
b. Only points 1, 2 and 3 are true.
c. Only points 2, 4 and 5 are true.
d. Only points 2 and 4 are true.
e. Only points 2, 3 and 5 are true.
1. It is a reductometric titration.
2. The cerium ion undergoes a change in
its oxidation number.
3. The oxidation number of cerium
increases.
4. The oxidation number of cerium
decreases.
86
25. Which statements are not true for atomic absorption spectroscopy?
1.
2.
3.
4.
5.
Tungsten and hollow cathode lamps can be used as light source.
Contamination originating from the die during tableting can be detected.
Any element can be measured in solution.
ICP can be used for the excitation of the atoms.
Individual light sources are necessary to the measurement of each element.
a.
b.
c.
d.
e.
All of the answers are false.
Only points 1, 2, 3 and 4 are false.
Only point 5 is false.
Only points 2, 3 and 5 are false.
Only points 3 and 4 are false.
26. Which statements are true?
1.
2.
3.
4.
5.
pH is directly proportional to the emf.
The absorbance depends on the length of the light path.
pH is inversely proportional to the emf.
The conductivity is independent of the surface of the electrode.
In determinations of the molar relative conductance, the conductances of the
individual ions are additive.
a.
b.
c.
d.
e.
Only points 1, 2 and 5 are true.
Only points 2, 3 and 4 are true.
Only points 1, 4 and 5 are true.
Only points 2, 3 and 5 are true.
Only points 1, 2 and 4 are true.
27. Which statements are false?
1.
2.
3.
4.
2 ppm is the same as 2 mg/l.
4 ppb is the same as 4 μg/l.
2.5 μm is the same as 400 cm-1.
1.0 absorbance is the same as 90%
transmittance.
5. 5 ppm is the same as 5 μg/l.
a.
b.
c.
d.
e.
Only points 2 and 3 are false.
Only points 1 and 4 are false.
Only points 3 and 4 are false.
Only points 2 and 4 are false.
Only points 4 and 5 are false.
28. According to the Pharmacopoeia Hung. VIII, which compounds are analyzed by direct
conductometry?
a.
b.
c.
d.
e.
Alkaline compounds.
Distilled water.
Benzoic acid.
Acetylsalicylic acid.
Acidic compounds.
29. Which effects shift the electromagnetic radiation absorption maximum of a compound
to higher-energy wavelengths?
a.
b.
c.
d.
A bathochromic shift.
A hypochromic shift.
A hypsochromic shift.
A hyperchromic shift.
87
30. What is the mathematical relationship between the conductance of a solution and the
distance between the electrodes?
a. The conductance of the solution is directly proportional to the distance between the
electrodes.
b. The conductance of the solution is inversely proportional to the distance between the
electrodes.
c. The conductance of the solution is directly proportional to the square of the distance
between the electrodes.
d. The conductance of the solution is directly proportional to the logarithm the distance
between the electrodes.
31. An alkaline solution is titrated with a standard acidic solution. Which of the following
statements are true for the titration curves?
a. A local minimum is observed at the equivalence point in the case of the first
derivative.
b. A local minimum is observed at the equivalence point in the case of the second
derivative.
c. The second derivative is zero at the equivalence point.
d. A local maximum is observed at the equivalence point in the case of the second
derivative.
e. A local maximum is observed at the equivalence point in the case of the first
derivative.
32. 0.10 g of substance is dissolved in 100 ml of methanol. 5 ml of 0.1 M Na2CO3 is added to
0.5 ml of the solution, which is diluted 100 ml. What blank solution should be prepared?
a.
b.
c.
d.
e.
Methanol is the blank solution.
0.1 M Na2CO3.
0.01 M Na2CO3.
0.02 M Na2CO3.
0.005 M Na2CO3.
33. Phenolphthalein is diluted with Na2CO3 solution. What effect can be observed?
a.
b.
c.
d.
A bathochromic shift.
A hypochromic shift.
A hypsochromic shift.
A hyperchromic shift.
34. How does the number of ions change after the equivalence point in the conductometric
titration of a weak acid and a weak base?
a. It increases.
b. It does not alter.
c. It decreases.
88
35. How does the number of the ions change before the equivalence point conductometric
titration of a strong acid and a strong base?
a. It increases.
b. It does not alter.
c. It decreases.
36. What kind of chemical reaction takes place between the peptide bonds in proteins and
the biuret reagent?
a.
b.
c.
d.
e.
Oxidation.
Reduction.
Complex formation.
Substitution.
Addition.
37. What is the first step in a photometric measurement?
a.
b.
c.
d.
Determination of the absorption of the sample.
Determination of the absorption of the calibrating solutions.
Determination of the absorption maximum of the sample.
Determination of the baseline.
38. How does the slope of a calibration curve change when less is measured of the
reference material compared with the given value?
a. The slope of the calibration curve increases.
b. The slope of the calibration curve does not change.
c. The slope of the calibration curve decreases.
39. What kind of light source can be applied in the wavelength region 380-780 nm?
a.
b.
c.
d.
A deuterium lamp.
A ceramic rod.
A tungsten lamp.
A halogen lamp.
40. Which statements are false for the titration of Unguentum ad vulnera?
a. Salicylic acid is titrated directly with standard NaOH solution.
b. The volume relating to H3BO3 is calculated by subtraction oftthe values at the first
inflexion point from the value at the second inflexion point.
c. The complexes of H3BO3 with polyols decrease the pH.
d. H3BO3 is titrated as a trivalent acid.
41. The conductance of Aqua purificata is:
a.
b.
c.
d.
0; it should not contain any contamination.
1.1 μS/cm.
3.4 μS/cm.
4.3 μS/cm.
89
42. Which statements are true?
1. Standard I2 solution is used for the titration of oxidative substances.
2. Standard I2 solution can be used for the titration of the antioxidant ascorbic acid.
3. During the titration of metamizole sodium, the resulting formaldehyde is oxidized to
formic acid by I2.
4. The end-point of Algopyrin inj. titration is indicated by the appearance of the blue
color of starch.
5. The sample is cooled during the titration of Algopyrin inj. because the resulting H2SO4
should not be warmed up.
a.
b.
c.
d.
e.
Only points 1, 2 and 3 are true.
Only points 1, 2, 3 and 4 are true.
Only points 3 and 5 are true.
Only points 2 and 4 are true.
All the answers are true.
43. Which statements are true for the retention time?
1.
2.
3.
4.
The retention time is the time required for the entire separation of a sample.
The retention time is different for all substances.
The retention time is the most important parameter of a chromatogram.
The retention time is the time between the application of the sample and its
appearance in its maximal concentration.
5. The retention time is also called the back-titration time.
a.
b.
c.
d.
e.
Only point 1 is true.
Only points 1, 2 and 3 are true.
All the answers are true.
Only points 2, 3 and 4 are true.
Only points 2, 3 and 5 are true.
44. Which statements are not true for atomic absorption spectroscopy?
1.
2.
3.
4.
5.
Tungsten and hollow cathode lamps can be used as light source.
Contamination originating from the die during tableting can be detected.
Any element can be measured in solution.
ICP can be used for the excitation of the atoms.
Individual light sources are necessary for the measurement of each individual element.
a.
b.
c.
d.
e.
All of them are false.
Only points 1, 2, 3 and 4 are false.
Only point 5 is false.
Only points 2, 3 and 5 are false.
Only points 3 and 4 are false.
45. How can an exact standard NaOH solution be prepared?
a. An exact NaOH standard solution cannot be prepared. It should be standardized before
use.
b. NaOH should be measured directly.
c. Through the direct measurement of NaOH in an inert gas atmosphere.
d. Through the direct measurement of NaOH at 0% humidity.
90
46. Which statements are true for the determination of protein concentration?
1. Both UV and VIS spectroscopy can be used.
2. The applied dyes shift the absorption maximum of the electromagnetic radiation
toward lower-energy wavelengths.
3. The oxidation of copper ions is utilized in several techniques.
4. Biuret or BCA reactions are good examples of protein concentration determination.
a.
b.
c.
d.
e.
Only points 1, 2 and 4 are true.
Only points 1, 2 and 3 are true.
Only points 2 and 3 are true.
Only points 2 and 4 are true.
Only points 2, 3 and 4 are true.
47. Which statements are not true for atomic absorption spectroscopy?
a.
b.
c.
d.
Tungsten and hollow cathode lamps can be used as light source.
The Pb contamination of the medicinal plants can be determined by this method.
Any element can be measured in solution.
Individual light sources are necessary for the measurement of each individual element.
48. Which statemens are not true?
a. Steroids can be quantitated by UV-VIS photometry after their condensation with
amines.
b. Picrate salts absorb UV light.
c. Acetylsalicylic acid absorbs UV light.
d. A quartz cell is used for measurements in the UV range.
49. What can disturb photometric measurements?
a.
b.
c.
d.
e.
Fingerprints on the wall of the cell.
Suspensions.
Solvent on the wall of the cell.
Bubbles in the sample solution.
All of the above.
50. What are the advantages of photometric measurements?
a.
b.
c.
d.
e.
Indicators can be excluded.
Dark solutions can be titrated.
Precipitation titration is possible.
They are more accurate than traditional classical titration.
All of the above.
91
APPENDIX
UNICAM UV/VIS SPECTROPHOTOMETER MANUAL
1. Turn the spectrophotometer on 15 min before the measurement. (The ON/OFF button
is on the left-hand side of the instrument.)
2. Recording a spectrum, and determination of the absorption maximum:
a. Scan, then ENTER.
b. Check the following parameters:
i. Mode: It should be ABS.
ii. Start: EDIT. Give the shorter wavelength of the scanning range, then
ENTER.
iii. Stop: EDIT. Give the longer wavelength of the scanning range, then
ENTER.
iv. Peak table: EDIT→PEAKS→ENTER.
3. Choose the appropriate measuring cell:
a. UV range: quartz cell.
b. VIS range: glass or plastic cell.
4. Determination of the absorption maximum:
a. Place the cell containing the reference solution into both slots of the
spectrophotometer. The light comes from the back to the front.
b. Push ZeroBase and record the baseline throughout the chosen spectral range.
c. Put the cell containing the third calibration solution into the sample slot. Keep
the reference solution in the reference slot.
d. Start the measurement with RUN.
e. View results: 5th gray button under the screen. Record the value of the
absorption maximum.
5. Go back to the main menu: HOME.
6. Choose Quant, then ENTER.
7. Determination of the calibration curve:
a. Set the correct wavelength:
i. Wavelength→EDIT→give the value of the absorption maximum,
then→ENTER.
b. Set the parameters of the calibration:
i. No Standards, number of calibration solutions: EDIT→5→ENTER.
ii. Standards (first gray button under the screen), then EDIT, give the
concentrations of the calibration solutions, then ENTER.
iii. Calibrate (third gray button under the screen), then ENTER.
iv. Place the first calibration solution into the sample cell, then RUN.
v. Repeat the measurement with all the other calibration solutions as in
point iv.
93
vi. View results: Check the equation of the calibration curve, and note the
value of the coefficient.
8. Determination of the sample concentration:
a. Place the sample into the sample cell, RUN.
94
UV-1601 SHIMADZU SPECTROPHOTOMETER MANUAL
1. Turn the spectrophotometer on 15 min before the measurement. (The ON/OFF button
is on the left-hand side of the instrument.)
2. Recording of the spectrum, and determination of the absorption maximum:
a. Choose Spectrum, 2 from the numerical characters.
b. Check the following parameters:
i. 1. Measurement mode: ABS.
ii. 2. Scanning range: give the appropriate wavelength range, depending
on your sample:
1. 2 from the numerical characters
2. Give the longer wavelength of the scanning range, then ENTER.
3. Give the shorter wavelength of the scanning range, then ENTER.
3. Choose the appropriate measuring cell:
a. UV range: quartz cell.
b. VIS range: glass or plastic cell.
4. Determination of the absorption maximum:
a. Place the cells containing the reference solution into both slots of the
spectrophotometer. The light comes from the left to the right.
b. Push the F1 button BaseCorr: for baseline correction throughout the chosen
wavelength range.
c. Place the cell containing the middle calibration solution into the closer sample
slot.
d. Start the measurement with the Start/Stop button.
e. Analyze the data with the DataProc F2 button.
f. To obtain the peak(s) at the absorption maximum, push button 3 Peak and note
the value(s).
5. Go back to the main menu:
a. RETURN
b. RETURN
c. RETURN
d. MODE.
e. Current data not saved.
f. OK→F3 button.
6. Choose: „Quantitation” button 3.
7. Determination of the calibration curve:
a. Set the correct wavelength:
i. Button 1
95
ii. Button 1 again
iii. Give the correct value, then ENTER.
b. Set the parameters of the calibration:
i. Button 2 Method
ii. Button 3 Multi-point calib.
1. No of Std. Number of standards: 5.
8. Determination of the calibration curve:
a. First push the Start/Stop key.
b. Give the concentrations of the calibration solutions. Push ENTER after each
value.
c. Button 2 Meas (only cell 1): place the first solution into the sample slot (the
closer one); the cell containing the reference solution is kept in the reference
slot, and the measurement is started with the Start/Stop key. Repeat this step
with all the calibration solutions.
d. Draw the calibration curve with F1 CalCurb.
e. Get the equation of the calibration curve with F4 and note the value of r2.
f. RETURN (twice).
9. Determination of the sample concentration:
a. F3 SamplMeas.
b. Start/Stop.
96
MARS CEM MICROWAVE DESTRUCTOR MANUAL
1. Place the sample into the container of the microwave destructor and add the appropriate
amount of acid. Strive to wash all the sample off the wall of the container.
2. Keep the blue valve open and place the lid on the container.
3. Close the blue valve, place the containers into the special holders and fix them.
4. Turn the machine on. (The button is at the right-hand side of the machine.)
5. Choose the appropriate program:
a. Load Method→Select
b. User directory→Select
c. GYAK-HP500→Select
6. Place the samples into the microwave destructor. N.B. Never start the program without
the reference container.
7. Green button→START
The program used by the microwave oven:
Time (min)
5
10
15
20
25
30
Power (%)
80
80
100
100
100
100
The table indicates the power as the percentage of the maximum 400 W. The
microwave oven is controlled by the pressure measured in the reference valve. The maximum
pressure used by the program is 80 psi (5.5 bar).
The powertime curve of the microwave oven
97
ATOMIC ABSORPTION SPECTROMETER MANUAL
1. Turn the compressor on AA1 in the preparation laboratory.
2. Check the pressure in the atomic absorption laboratory on AA2 and wait until it reaches
4 bar.
3. Open the acetylene gas valve on the top (yellow: AA3). Check the pressure in the
cylinder (the left indicator should not go below 3 bar) and check that the pressure goes
to the spectrometer (the right indicator should not go above 1.5 bar).
4. Turn on the extractor hood.
5. Turn on the atomic absorption spectrometer: AA4.
6. Turn on the computer: AA5.
7. Turn on the printer: (AA6).
8. Click on the AA INST.exe icon. Choose the FLAME method. Wait until the
background of the 4100 icon at the bottom of the screen turns dark-blue. Choose
MANUAL.
9. Choose the experimental element: step 1: Defaults, step 2: choose the correct element
and step 3: OK.
10. Click on the Windows menu, choose the Align lamps function and set the correct
current intensity for the lamp. (The parameters can be found on the lamps.)
11. Set the energy to the maximum with the white screw on the lamp and accept it by
clicking AGC/AIC.
12. Enter the Flame Control menu, and click on the Flame icon to ignite the flame. Check
the gas ratio, which should be: C2H2: 2,5; air: 8,0.
13. Enter the Windows menu again, click on Element parameter and then on Instr. And
check:
a. Set Slit to 0.7 (Height High).
b. Set the time of the measument to 5 s.
c. Set the BOC to 2 s.
d. Set the Read delay to 2 s.
e. Repeat the sample and the standards twice.
f. The Burner move should be in the ON position.
14. Enter the Element Parameter and choose the Calib menu.
a. The first calibration solution is the reference solution.
b. Set the standards as st1, etc.
c. The second column contains the concentrations of the calibration solutions.
Indicate them to at least three decimals.
15. Enter the Windows menu, and choose ID Weight parameter. Give the sample
parameters.
16. Choose Flame Control again. Click on Find Reference and put the capillary into the
reference solution, then into st2 according to the commands.
98
17. Enter the Windows menu, choose Continous graphics and put the capillary into the
reference solution. Push key F3 to set zero absorbance. Put the capillary into st2 and set
the absorbance influencing the suction. (Here the 0.3 mg/l standard solution is used and
the absorbance in this case should be about 0.182; ±20% difference is acceptable.)
18. Enter the Calibration menu.
a. Put the capillary into the reference solution, and click on autozero. Repeat this
measurement twice to stabilize the zero value.
b. Measure the absorbances of the standard solutions. Wash the capillary with
distilled water before each measurement.
c. Check the value of the coefficient after the calibration. The closer the value is to
1 is the better the calibration curve.
d. Print the calibration curve File menu, then Print Image.
19. Put the capillary into the sample. Start the measurement by pushing F4. The
spectrometer measures the samples twice. Wash the capillary with distilled water before
each sample. The results are recorded and printed out.
20. Turning off:
a. Close the gas cylinder (wait until the indicators show zero)
b. Enter the File menu and then Quit to Desktop
c. Turn off the spectrometer: AA4
d. Turn off the extractor hood
e. Turn off the printer: AA6
f. Enter the File menu and then Exit to DOS
g. Turn off the compressor: AA1
99
HPLC MANUAL
How to start up the HPLC
The in-line filters should be dried with a paper tissue and then placed into the
appropriate solution:
Solvent line A: 50 mM phosphate buffer pH 6.3.
Solvent line B: acetonitrile.
Turning on the (SHIMADZU Prominence UHPLC system) instrument:
1. the pump (LC-20AD) (N.B. the degasser is turned on together with the pump
and initially gives an error signal);
2. the thermostat (CTO-20A);
3. the detector (SPD-M20A); wait 2 min to allow the vacuum pump of the degasser
(DGU-20ASR) to ensure the optimal vacuum.
Preparation of the analysis program with the LC Solution software
Double click on the blue icon of LC Solution. The LC Solution Launcher starts. Click
on the Operation option in the blue window, where the Analysis Series HPLC 1 icon should
be selected. A pop-up window appears. User ID: Admin, Password: leave empty, and then OK.
Two whistles are heard and a gray window will show: Connecting the LC Instrument… An
application appears in the Data Acquisition window: LC Real Time Analysis. Wait until the
LC, PDA connection is switched to Ready (green background).
Now prepare the project folder: Choose the File menu, Select Project (Folder)...; the
Project (Folder) Selection window appears, where the Panadol próba folder should be
selected. The program marks the folder in gray color. Click on New Folder: the Create New
Folder window appears; write the name of the folder, e.g. “Moday morning 2015” into the line
of Please input new folder name. Choose the new folder with a single click in the Project
(Folder) Selection window (gray background). Close the dialog window.
Creating method file:
1. File menu, Open Method File.
2. A dialog window pops up. Location: Choose the Panadol próba folder and
then the Hallgató folder. Double click on the mérés gradiens method file. Save
the opened method under a new name.
3. File menu, Save Method File As...: type the name of the method file (e.g.
practice-Monday), then Save.
Description of the HPLC method.
The conditions of the analysis are to be seen in the Instrument Parameters View
window. N.B. Here these parameters can be modified, but not now! Click on the Normal
button and the Simple Settings option shows the duration of the analysis (Time Program, LC
Stop Time: 5.00 min). The PDA is active; the end of the data acquisition is also 5 min (End
Time: 5.00 min). Information on the pumps is included here. Pumps: Mode: Low pressure
gradient. This means that the analysis is performed in gradient mode; the instrument mixes
the solvents according to the program, which changes linearly in time. At present the Total
Pump A Flow: 0.000 ml/min, and concentration of Solvent B: 10.0%. The gradient program is
as follows:
100
Time
Module
Action
Value
1.
0.01
Pumps
Concentration of B
10
2.
4.00
Pumps
Concentration of B
20
3.
4.10
Pumps
Concentration of B
10
4.
5.01
Stop
This means that the initial 10% acetonitrile concentration increases to 20% by the 4th
min and then changes back to 10% in 0.1 min. By using a gradient and gradually increasing
the proportion of the organic component, a shorter retention time can be achieved. Further, the
wide peaks resulting in isocratic elution can be reduced by using gradient elution.
By clicking on the Normal mode LC Time Prog. option, information becomes
available about the PDA (PhotoDiode Array). Lamp: D2 & W means that deuterium and
tungsten lamps are used. Wavelength: Start wavelength: 268 nm; End wavelength: 272 nm.
270 nm is used for data acquisition, but it is worth having a wavelength interval.
Full spectra (190-800 nm) should not be measured, because extremely large data files
with very much unnecessary information would be created. The optimal wavelength is
determined in advance: 270 nm. Paracetamol has an absorption maximum at 238 nm, but the
measurement is performed at 270 nm, i.e. the absorption maximum of caffeine.
The spectrum of Paracetamol in the interval 200-400 nm
The spectrum of caffeine in the interval 200-400 nm
The sample contains less caffeine than paracetamol (approximately one-eighth of its
amount). The diluted sample contains trace only amounts of caffeine, and therefore you have
to set the wavelength to the absorption maximum of caffeine to determine the concentration of
101
the active ingredients correctly. Paracetamol absorbs UV light at 270 nm with sufficient
intensity (~700 mAU), and this wavelength is therefore optimal for the detection during the
HPLC analysis.
Preparation of the HPLC system for the measurement:
1.
Purge: removal of bubbles from the system with high flow speed. The valve to
the column is closed.
The purge valve in the pump unit should be opened by turning the knob in the
anticlockwise direction twice (the first turn is difficult).
The value of Solvent B Conc. should be changed from 10% to 50% in the Normal
mode Simple Setting option to degas both of the solvent lines. The value of Total Pump A
Flow should be altered to 5 ml/min. (N.B. Only when the purge valve is open!) Click on
DownLoad. A dialog window pops up: Current Method File will be Saved. OK. The Oven
ON/OFF and the Pump ON/OFF should now be ON with a single click on the appropriate
buttons on the instrument control panel. Leave the system in operation mode for 2 min after
the high flow has started. Two minutes later, the flow rate should be zero again: Total Pump
A Flow: 0 ml/min, DownLoad, OK. Close the purge valve. The Concentration of Solvent
B should be 10% again (DownLoad, OK).
2.
Increasing the flow rate gradually
One of the goals is to wash the column with the solvent and the other is to reach the
flow rate used during the measurement.
It is advisable to alter the flow rate gradually to protect the column. The pressure
increases when the flow rate is increased, and the stationary phase might therefore be damaged
if the highest flow rate is used immediately. Small increments are applied. The flow rate should
be increased in 0.2-ml/min steps until the final 1.5 ml/min is reached. All steps should be held
for 2 min to stabilize the pressure. The following values are set: 0.2, 0.4, 0.6, 0.8, 1.0, 1.3,
1.5 ml/min. N.B. The program accepts only decimal points. The values of the pressure and
the flow rate can be followed in the data acquisition window. The uppermost yellow area shows
the changes in pressure and intensity with time. The window can be widened if necessary.
Currently it is 5 min wide. Click the right-hand button on the yellow area and choose Display
Settings, General option in the pop-up window. The time in the Time Range should be
overwritten to 60 min then OK. The steps are now visible.
Check the second window if 270 nm is indicated. If not, overwrite the value. Click the
right-hand button on the yellow area and choose Display Settings, PDA option. Overwrite the
value of the wavelength to 270 nm in the first field (Wavelength), then OK.
3.
Washing the column
The column should be washed for an additional 30 min after the final 1.5 ml/min flow
rate has been reached. This step is necessary to equilibrate the column and to reach a dynamic
balance between the stationary and mobile phases. Meanwhile, the Batch file should be
prepared.
4.
Preparation of the Batch file
The measurementmust now be planned. The program works according to the samples;
it evaluates them on the basis of the parameters specified here. The options of the analysis were
specified earlier (in the Method file). The program applies these in the case of each sample.
Click on the Batch Processing icon on the left side of the screen (additional tool bar). Batch
102
view becomes visible. File menu, New Batch File. The actual batch file can be prepared in the
empty table.
File menu, Save Batch File As… Write the chosen name (e.g. practice-Monday), OK.
Data should only be entered into the following columns: Sample Name, Sample Type,
Analysis Type, Method File, Data File, Level, Inj. Volume.
Fill in the table according to the example below:
103
Sample Name: Enter the name of the samples. First a blank solution is injected
(solvent), and then the standards for the calibration routine. Standard 1 will be injected 5 times.
Name the standards, for example s11, s12..., Standard 2 then will be injected 2 times to
determine the correlation. Finally enter the name of the samples, e.g. the name of the student.
Sample Type: the blank remains unknown 0: Unknown. The next sample is the
Standard1 solution, injection 1. This is the first calibration solution. Click on the arrow in the
right corner of the area. Indicate the sample as Standard in the pop-up window, then choose
the Initialize Calibration Curve among the calibration types, OK. In the Sample type,
Standard is indicated and “I” in brackets as Initialize.
The next sample is the Standard1 solution, injection 2. Scroll down the arrow. This
sample is also a Standard, but it is not initial, so click on Add Calibration Level icon and
then OK. The sample has now been added to the calibration series, but not as an initial one. It
is indicated as a Standard only. This step is repeated with Standard1 to reach sample 5. The
calibration solutions of Standard2 and the type of the samples remain unknown, 0: Unknown.
Analysis Type: everything remains IT QT (IT: Quantitative Integration, QT:
Quantitative Calculation).
Method file: the name of the previously saved file appears in the first line if everything
was properly done. All the other lines should be filled by scrolling down the arrow and
choosing the appropriate method file. N.B. It is not possible to copy/paste the options. Be
careful to select the appropriate method file and check that the extension is .lcm.
Data File: the lines should be filled in individually. Click on the right-hand arrow and
the Select Data File window opens. Write the name of the file.Iit is advised to use the same
name as the name of the sample. Save. “.lcd” extension is added to the data file. Be careful: if
the line is filled, the data file will not be created with “lcd” extension and it cannot be saved.
Level#: remains 1. All lines should be filled in individually.
Inj. Volume: 20 should be indicated here as the injected volume from the loop onto
the column. The loop used by the instrument is 20 µl. This is the volume that reaches the
column from each sample. The optimal circumstances of the quantitative determination are
now defined.
Save the batch file when all the boxes are filled. Click on the Floppy/Save icon on
the toolbar.
Click on the Data Acquisition option below again. Check the washing program.
Prepare for the sample injections. It is important to inject the samples one after an other.
When one sample is completed (5 min), do not hesitate, but inject the next one. It is
important to inject the samples rapidly with the same time intervals in the case of gradient
elution. It is advisable to prepare the next sample while the previous one is running. The
sample loop cannot be filled with the sample while the sample is running.
5.
Injection of the sample
The instrument is supplemented with a Rheodyne manual injector. The samples are
injected manually with a Hamilton pipette. The Hamilton #825 microliter pipette is used during
the practicals. The pipette can hold 250 µl, and the tip is replaceable. The tip is a blunt-end tip.
The pipette is designed to guarantee a firm grip. The pipette should be rinsed before each
injection. You may begin with the blank solution. Approximately 250 µl of solution should be
sucked by any pipette slowly so as to avoid bubble formation. The tip of the pipette should be
elevated and the bubbles should be removed before injection. It is advisable to use a paper
tissue to prevent the solution spraying out. The bubble-free solution (approx. 200 µl) should
be emptied into the waste bottle. After the pipette has been washed, another aliquot is sucked
up and bubbles are removed if necessary. The volume of the solution should be at least 200 µl.
The loop too should be washed. The volume of the loop is 20 µl, so the 200 µl of solution in
the pipette is enough for the washing. The tip of the pipette should be placed into the injection
valve and pushed until it stops. The injector valve is in the load position. The sample is injected
slowly into the loop. If the process is correct, no solution appears at the tip, but the excess
amount overflows to the waste. If solution appears at the tip, the whole process should be
repeated. After a 30-min washing at 1.5 ml/min, the analysis can be started.
6.
Analysis
The first sample (the blank solution) is now in the loop. Switch back to the Batch table
in the software. Click on the Batch start in the supplementary tool-bar to start the analysis.
The screen now shows the analysis view. In the upper part of the screen, the actual view of the
Batch table is visible. The sample that is running at the moment is highlighted in gray; all the
others are yellow. Check the uppermost yellow area, which shows the changes in pressure and
intensity over time. Narrow the window if necessary to be 5 min wide. Click the right-button
on the yellow area and choose Display Settings, General option in the pop-up window. The
time should be overwritten in the Time Range to 5 min then OK.
The following two steps should be executed without any delay. A pop-up window
appears: Data Acquisition Start. Click on the Start button, and then immediately turn the
valve into the Inject position with a direct movement. After this, 20 µl of sample is injected
into the column. The solvent flows continuously through the column and the separation starts.
The middle window shows the chromatogram (green line). When the blank solution is
running, no peaks appear. For all of the other samples, two peaks are expected after the void
volume: first the paracetamol peak, and later the caffeine peak.
The next sample is prepared during the run. The Batch view always shows the next
sample. Now wash the pipette with Standard1 solution and then prepare the 200 µl of sample
for injection. This preparation allows you to inject the new sample immediately after the
previous sample. The Batch table switches to the next line when a sample is completed, and
the Data Acquisition window pops-up, asking for the next sample. The injection valve should
be turned back to the Load position. Inject the sample. Click on the Start button on the screen.
The injection valve is turned to the Inject position again. These steps should be alternated until
the last sample has been run. After the analysis is finished and all the samples show two peaks
(but not the blank solution), the instrument should be stopped. The flow rate should be changed
to 0 ml/min. Wait until the pressure becomes 0. Then turn off the instrument in the following
order: detector, thermostat, pump.
7.
Evaluation of the data
The next step is the analysis of the chromatograms. You need to know the area under
the curve to determine the concentration of the active ingredient. The program identifies the
peaks on the basis of their retention times. To determine the peak areas, the peaks should be
assigned, and the integration parameters should be set. This setup was completed previously.
Go back to the LC Solution Launcher. Start the Postrun application. The LC Postrun
Analysis view appears. Project in: Select Browse Folder... open the yellow folder and choose
the newly created folder; double click in Project(Folder) Selection. The window can now be
closed. The data from the measurement are uploaded and the data files become visible. You
may begin with the standards. Double click on the Standard1 1 file to open the data. New
windows will pop up in several views. The chromatogram will be visible in the
Chromatogram view. The calibration curve can be seen below. The values of Compound
Table View are used for the analysis. The table contains the data relating to the peaks of
106
Paracetamol
Name of the sample
Retention time
Area
Retention time
Area
S11
S12
S13
S14
S15
S21
S22
Caffeine
Name of the sample
S11
S12
S13
S14
S15
S21
S22
paracetamol and the caffeine. Click on the Results. Two data are necessary and those should
be registered into the table above (Ret. Time, and Area). You should record all the values of
the standards and the results of your own samples.
Close Postrun view.
8.
Determination of the active ingredient content of the sample; checking the
calibration in the Excel table
Double click on the (Eredmények) Results icon on the desktop. Copy the Minta Sheet
to the end of the series and rename the sheet, e.g. English-Monday. This new datasheet is used
to evaluate the results.
Data must be input into the active ingredient content calculation table. Be careful to
enter data only into the light-blue cells. The other cells contain equations and if these are
changed, incorrect results will be calculated.
107
The paracetamol peak areas of the S1 injections should be written into cells C7-C11 of
the table. The caffeine peak areas should be written into cells C29-C33 of the table. The
calculated Standard1 paracetamol concentration should be entered into cell D7, and the
caffeine concentration into cell D29.
The paracetamol peak areas of the Standard2 injections should be written into C20 and
C21, and the caffeine Standard2 areas into G28 and G29. The calculated concentrations of
these injections should be written into cells D20 and H28. Write the data only into the cells
mentioned above.
The input of the data on the samples starts with the peak areas of the chromatograms.
The peak areas of the samples of paracetamol and caffeine should be written into the table. The
next column contains the Labeled Claim/LC value in mg for the analyzed active ingredient.
The weights of the samples should be written into the next column, with four-decimal
accuracy. Be careful to give the same weights for both active ingredients as the same sample
was used in both analyses.
The reference (Average tablet weight in g) value should remain at 0.697, the value was
used in the calculations.
The following data can be found in the calculated cells:
RSD%: the relative percentage of the average peak area deviation of the standard1
injections. If this is below 2.0%, the deviation of the injections is appropriate. If it is higher
than 2.0%, discuss the situation with the tutor to find out what the problem is.
The parameters of the calibration curve are in cells B13-C15 and B35-C37. To calculate
the slope, these values will be used and the intersection with the axis should be zero.
Cells C23 and G31 show the correlation value and indicate wherter the calibration is
correct. If the absolute value is higher than 2.0 in any of these cells. Discuss the situation with
the tutor. The value is zero in the ideal case when the standard2 concentration calculated from
the calibration is equal to the theoretical concentration (based on the weight of the sample).
Column L contains the concentration of the active ingredient, based on the calibration
curve.
Column M contains the mass percentage of the active ingredient in one tablet.
Column N contains the amount of the active ingredient in the tablet in mg.
The individual printed out results should be shown to the tutor.
108
NMR SPECTRA
THE NMR SPECTRA WERE PROVIDED BY:
DR. HABIL. PÉTER FORGÓ
170
160
150
140
10
13
130
9
120
110
8
100
110
7
90
6
80
5
70
60
4
50
40
20.90180
11
39.73320
39.59150
39.45160
39.31150
39.17440
12
3.03
1.03
1.01
1.02
1.00
0.71
O
126.13880
124.16330
123.85840
13
133.85780
131.49730
150.31440
165.75820
169.30910
3
30
2.2461
2.4996
7.9618
7.9489
7.6308
7.6180
7.6050
7.3756
7.3630
7.3503
7.1952
7.1818
1
H NMR SPECTRUM OF ACETYLSALICYLIC ACID
acid acetylsal. dmso - 600
O
OH
O
CH3
ppm
C NMR SPECTRUM OF ACETYLSALICYLIC ACID
acid acetylsal. dmso 600
ppm
160
150
140
130
N
O
6.0
13
120
5.5
110
100
N
CH3
5. 0
90
111
4. 5
80
4.0
70
60
3.5
50
40
3.3588
7.4719
7.4590
7.4461
7.3439
7.3312
7.3305
7.2739
7.2615
7.2493
3. 0
30
2.98
2.5
20
2.1671
2.4999
2.6555
N
6.01
CH3
2.9064
CH3
2.98
1.08
2.00
1.98
1.00
H3C
9.98560
6. 5
43.47180
39.73170
39.59070
39.45150
39.31210
39.17360
39.03190
36.36210
125.51310
122.68960
122.20010
7.0
128.82390
7.5
135.25810
150.76490
162.65600
1
H NMR SPECTRUM OF AMINOPHENAZONE
aminofenazon dmso - 600
ppm
C NMR SPECTRUM OF AMINOPHENAZONE
aminofenazon dmso - 600
ppm
1
H NMR SPECTRUM OF BENZOIC ACID
7.9805
7.9794
7.9673
7.9653
7.5708
7.5585
7.5461
7.4644
7.4513
7.4386
2.03
1.00
2.06
benzoesav dmso - 600
O
0.75
OH
13. 0
12. 5
12. 0
11. 5
13
11. 0
10. 5
10. 0
9. 5
9. 0
8.5
8.0
7.5
ppm
C NMR SPECTRUM OF BENZOIC ACID
170
165
160
155
150
145
112
140
135
130
128.62750
129.46220
130.97390
132.91050
167.56480
benzoesav dmso 600
ppm
150
140
130
6. 5
13
120
110
6. 0
100
5. 5
90
5. 0
80
113
4. 5
70
4. 0
60
50
40
14.19420
7. 0
39.72610
39.58470
39.44500
39.30670
39.16760
N
4.3644
4.3556
4.3470
5.0507
5.0415
5.0323
7.9949
3.24
2.4999
2.4536
3.4516
3.4482
OH
0.37
NO2
3.7072
3.6985
3.6894
3.6808
N
2.19
2.19
1.09
1.00
H3C
48.29270
7. 5
59.81160
132.91650
8. 0
138.40770
151.96050
1
H NMR SPECTRUM OF METRODINAZOL
metrodinazol dmso - 600
3. 5
3. 0
30
ppm
C NMR SPECTRUM OF METRODINAZOL
metrodinazol dmso 600
20
ppm
160
150
140
6. 0
130
120
5. 5
110
100
5. 0
90
114
4. 5
80
70
4. 0
60
3. 5
50
40
10.81030
6. 5
35.81670
N
3. 0
30
2.97
2.97
3.00
1.99
H3C
49.28530
49.14360
49.00160
48.85980
48.71710
43.26070
2.00
2.97
2.01
H3C
73.14920
13
121.73290
7. 0
130.31620
128.57620
126.27460
7. 5
135.69370
153.01930
164.75230
2.3651
3.0668
3.0199
3.3124
3.3100
4.1161
4.8117
7.5242
7.5114
7.4980
7.3874
7.3823
7.3792
7.3738
1
H NMR SPECTRUM OF NORAMINOPHENAZONE
noramino-phenazon meod - 600
SO 3Na
N
CH3
N
O
2. 5
20
ppm
C NMR SPECTRUM OF NORAMINOPHENAZONE
noramino-phenazon meod 600
ppm
160
150
140
130
120
110
6. 5
6.0
100
115
5.5
90
80
5. 0
70
60
36.37740
N
O
CH3
CH3
50
4.9904
6.9460
6.9430
6.9323
6.9292
6.7509
6.7371
7.3273
7.3177
7.2433
7.2401
7.6365
7.9007
7.8899
8.2620
8.2512
3.8448
3.7989
O
3.01
3.06
O
4.1128
4.0448
H3C
3.03
3.04
1.99
O
56.77260
56.44910
56.16130
55.73500
77.21150
76.99940
76.78810
13
105.92520
104.84030
7. 0
1.01
1.01
1.16
1.01
1.00
1.01
H3C
112.37760
111.19730
7.5
122.38170
121.44990
120.69330
129.14420
127.91900
8.0
136.80130
149.34970
148.21720
153.93940
152.45180
156.86450
1.00
1
H NMR SPECTRUM OF PAPAVERINE
papaverin cdcl3 - 600
4. 5
4. 0
40
ppm
C NMR SPECTRUM OF PAPAVERINE
papaverin cdcl3 600
30
ppm
1
H NMR SPECTRUM OF PARACETAMOL
NH
1.9911
2.5005
3.5654
6.7074
6.6940
7.3647
7.3512
9.1579
9.6582
paracetamol dmso - 600
CH3
O
9. 5
9. 0
8.5
8.0
7. 5
13
7. 0
6. 5
6. 0
5.5
5. 0
4. 5
4. 0
3.05
0.46
2.06
2.04
1.02
1.00
HO
3. 5
3.0
2. 5
2. 0 ppm
C NMR SPECTRUM OF PARACETAMOL
160
150
140
130
120
110
100
90
116
80
70
60
50
40
23.75170
39.73080
39.59000
39.45080
39.31230
115.07970
121.00010
131.04160
153.22760
167.70020
paracetamol dmso 600
30
ppm
160
150
140
130
120
110
100
90
117
80
70
3. 5
60
3. 0
50
2. 5
40
30
19.21570
4. 0
25.11760
4. 5
28.24710
5. 0
38.33740
5. 5
57.59350
57.51060
55.35100
49.28150
49.14060
48.99940
48.85650
48.71490
45.14760
6. 0
61.28300
13
1.02
2.03
0.96
1.03
0.95
2.18
2.02
3.04
1.02
0.98
1.10
0.95
0.93
1.01
1.01
1.00
0.97
HO
67.90120
6. 5
102.07470
117.09970
7. 0
120.47900
123.85860
7. 5
127.36100
8. 0
131.48420
139.24550
8. 5
148.04980
147.96780
147.10910
144.56570
160.15030
1.00
8.680
7.910
7.895
7.795
7.787
7.487
7.483
7.379
7.375
7.364
7.360
6.302
5.764
5.753
5.747
5.735
5.724
5.718
5.707
5.107
5.079
5.001
4.983
4.895
4.309
4.025
3.631
3.614
3.610
3.603
3.592
3.313
3.310
3.307
3.305
3.300
3.288
3.283
3.279
3.270
3.266
3.261
3.257
2.819
2.246
2.234
2.224
2.211
2.203
2.197
2.191
2.083
2.078
1.966
1.511
1.506
1
H NMR SPECTRUM OF QUININE
kinin meod - 600
H2C
N
H3C
O
2. 0
1. 5
20
ppm
kinin meod 600
C NMR SPECTRUM OF QUININE
ppm
1
H NMR SPECTRUM OF SULFADIMIDINE
11. 0
10. 5
10. 0
9. 5
9. 0
8. 5
13
8. 0
7. 5
7. 0
6. 5
2.2140
6.03
3.4610
5.9610
1.98
6.6517
6.6043
6.5899
1.01
2.02
2.00
0.90
7.6939
7.6795
11.0647
sulfadimidin dmso - 600
6. 0
5. 5
5. 0
4. 5
4. 0
3. 5
3. 0
2. 5
ppm
C NMR SPECTRUM OF SULFADIMIDINE
O
O
23.07260
39.86910
39.72970
39.59080
39.45180
39.31220
39.17300
CH3
113.74380
112.40110
111.92250
111.40600
125.10320
130.37300
152.90540
156.65140
167.31950
sulfadimidin dmso - 600
N
S
NH
N
CH3
H2N
160
150
140
130
120
110
100
90
118
80
70
60
50
40
30
ppm
119